A novel variant of the RON receptor tyrosine kinase derived from colorectal carcinoma cells which lacks tyrosine phosphorylation but induces cell migration

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journal homepage: www.elsevier.com/locate/yexcr

Research Article

A novel variant of the RON receptor tyrosine kinase derived from colorectal carcinoma cells which lacks tyrosine phosphorylation but induces cell migration Da Wanga,b,1, Wei-Feng Laoa,b,1, Ye-Ye Kuangb, Shu-Min Gengb, Li-Juan Mob, Chao Hea,b,n a

Department of Colorectal Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine/SRRSH, 3 East Qingchun Road, Hangzhou, Zhejiang 310016, People’s Republic of China b Biomedical Center, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China

article information

abstract

Article Chronology:

Generation of splice variants in the RON receptor tyrosine kinase facilitates the invasive

Received 17 April 2012

phenotype of colorectal cancers. Here, we report a new splice variant of RON in the human

Received in revised form

colorectal cancer cell line HCT116. This variant is encoded by a transcript differing from the full-

23 August 2012

length RON mRNA by an in-frame deletion of 106 amino acids in the extracellular domain of

Accepted 28 August 2012

RON b-chain. The deleted transcript originates by an alternative deletion of exon 2 and exon 3.

Available online 7 September 2012

The molecular weight of this variant is 160 kDa. Thus, we named this variant ROND160E2E3. This

Keywords:

variant is a single-chain protein and expressed in the intracellular compartment. We found that

Splicing variants

ROND160E2E3 had no tyrosine phosphorylation ability, but it has constitutively activated

Receptor tyrosine kinase

Akt activity in transfected HEK293 epithelial cells. The expression of this variant in HEK293

Cell movement

cells resulted in an increased migratory activity in vitro mediated through the PI-3K/Akt

PI-3K/Akt pathway

pathway. Our data describes a new splice variant of RON and suggests a novel role for the RON receptor in the progression of metastasis in colorectal cancer. & 2012 Elsevier Inc. All rights reserved.

Introduction Receptor-type protein tyrosine kinases (RPTK) are a group of cell surface proteins that play an essential role in regulating cell growth, differentiation and survival [1]. There are 17 families of RPTK that have been identified [1]. Among them, the receptors for epidermal growth factor (EGF) [2], hepatocyte growth factor (HGF)/scatter factor (SF) [3] and nerve growth factor [4] are

typical examples of RPTK. One example of RPTK is MET, the receptor for the hepatocyte growth factor receptor (HGF), which plays an important role in the tumorigenesis and invasiveness of certain epithelial carcinomas [5]. The MET family includes MET, C-Sea and RON [6–8]. Studies have indicated that C-Sea might be the chicken homolog of human RON [9]. The recepteur d’origine nantais (RON) receptor tyrosine kinase, also known as the macrophage stimulating protein

Abbreviations: DMEM, Dulbecco’s modified eagle medium; EGF, epidermal growth factor; HGF, hepatocyte growth factor; MSP, macrophage stimulating protein; RPTK, receptor-type protein tyrosine kinases; RT, reverse transcription n

Corresponding author at: Department of Colorectal Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine/SRRSH, 3 East Qingchun Road, Hangzhou, Zhejiang 310016, People’s Republic of China. Fax: þ86 571 86960497. E-mail address: [email protected] (C. He). 1 Da Wang and Wei-Feng Lao contributed equally to this study 0014-4827/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2012.08.006

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(MSP) receptor [10], is another member of the MET protooncogene family [5]. The cDNA encoding human RON was cloned from a human foreskin keratinocyte cDNA library [7]. The mature RON is a 180-kDa heterodimer composed of a 40-kDa a-chain and a 150-kDa transmembrane b-chain with intrinsic tyrosine kinase activity [7]. The RON gene contains 20 exons and resides in chromosome 3p21 [11]. Studies have shown that RON is important for embryological development [12]. The activation of RON mediates multiple signaling cascades that are involved in cell motility, adhesion, proliferation, and apoptosis [13,14]. The induction of RON phosphorylation can be achieved through ligand-dependent and independent manners [15]. The ligand for RON was identified as macrophage stimulating protein (MSP) [10], which is also known as HGF-like protein [16]. The generation of splice variants is one of the mechanisms of RON activation in cells that contain RON variants. Six variants have been identified since the first variant, ROND165, was found in the stomach cancer cell line KATO III [15,17–22]. Four of the six variants have the ability to induce a motile and invasive phenotype in epithelial cells [17,20]. The other two variants can inhibit tumorigenic activities mediated by RON activation [18,19,21,22]. In this paper, we reported a new RON variant derived from the colon cancer cell line HCT-116. In this variant, exon 2 and exon 3, which encode 106 amino acids in the extracellular domain of the RON b chain, are deleted; the molecular weight of this variant is 160 kDa. Thus, we named this variant ROND160E2E3. We found that ROND160E2E3 had lost its tyrosine phosphorylation ability but showed constitutively activated Akt activities in transfected HEK293 epithelial cells. We also studied the function of this variant and found that expression of ROND160E2E3 correlates positively with enhanced motile activities in vitro.

Materials and methods Cells and reagents The human colorectal carcinoma cell lines colo320, HCT116, DLD1, HT29, RKO, SW620, SW480 and the human embryonic kidney cell line HEK293 were from American Type Culture Collection (Rockville, MD, USA). The human cecum undifferentiated adenocarcinoma cell line (HCe-8693) was a gift by Dr. Zhu Y.P. (Zhejiang Cancer Hospital, China). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal calf serum and maintained at 37 1C in a humidified atmosphere with 5% CO2. Recombinant human MSP was from R&D Systems (Minneapolis, MN, USA). Rabbit IgG antibodies specific to human RON C-terminal peptides were used as previously described [23]. The mouse anti-phospho tyrosine mAb (p-Tyr-100), rabbit IgG antibodies specific to regular or phospho Erk1/2 (p44/42), and the p-Akt antibody (Thr308) were from Cell Signaling, Inc. (Beverly, MA, USA). Rabbit IgG antibodies specific to the extracellular domain of human RON (H160) and the C-terminal of RON (C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The PI-3K inhibitor LY294002 was from Calbiochem (San Diego, CA, USA). The mouse monoclonal antibody against the RON a-chain was from BD Biosciences (San Jose, CA, USA).

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Tyrosine kinase assay kit (non-radioactive) was from Upstate (Lake Placid, NY).

Tissue specimens Colorectal cancer tissue specimens were obtained from patients operated in the Department of Colorectal Surgery, SRRSH. All patients consented to the use of their tissue for research. Tissues were stored in liquid nitrogen.

Immunoprecipitation and Western blotting The cells (35  106 per sample) were lysed with 200 ml of lysis buffer (50 mM Tris, pH 7.4, 0.5% Triton X-100, 0.5% NP-40, 150 mM NaCl, 2 mM EDTA, 100 mM vanadate, 5 mg/ml leupeptin, 5 mg/ml aprotinin and 10 mg/ml soybean trypsin inhibitor). Cellular proteins were incubated overnight at 4 1C with 2 mg of C-20 coupled to protein A/G plus agarose beads (Thermo Fisher Scientific, Rockford, IL, USA). Samples were separated on a 7.5% polyacrylamide gel under reducing conditions and transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA). Western blotting was performed using the rabbit IgG to RON or the mAb p-Tyr-100 to phosphotyrosine followed by goat anti-mouse or rabbit IgG conjugated to horseradish peroxidase (Roche). The reaction was developed with ECL reagents and quantitated using the BandScan densitometry software. In some experiments, the membrane was treated with stripping buffer and re-probed with other antibodies.

In vitro tyrosine kinase assay To test the tyrosine kinase activity of RON and its variant, cells were placed in serum-free media for 12 h and then treated with 2 nM MSP for 5 min. Cells were washed with cold 1X PBS, total cellular protein was extracted with lysis buffer and immunoprecipitation was performed using rabbit IgG antibodies specific to the C-terminal of RON (C-20) and protein A/G plus agarose. The isolated immune complex was incubated with a biotinylated substrate peptide containing repeats of poly Glu4–Tyr bound to streptavidin-coated 96 well plates and Millipore tyrosine kinase assay kit 17–315 was used to measure kinase activity.

Reverse transcription (RT)-PCR and DNA sequencing Total RNA was isolated from HCT116 cells using TRIzol (Life Technologies). Reverse transcription was performed using 2 mg of total RNA with a SuperScript Preamplification Kit (Life Technologies). PCR was conducted on the products of RT reactions. The oligomers for the PCR amplification used to clone the ROND160E2E3 cDNA were designed on the basis of the RON cDNA sequences: pair 1, sense oligomer, nt 183 to 164 (50 -GAGGGCCGGGAAGGGATTTG30 ) and antisense oligomer, nt 394 to 415 (50 -TGCTGGTGCTGGATCCCGCGCT-30 ); pair 2, sense oligomer, nt 48 to 66 (50 -TGCCTCAGTCCTTCCTGTT-30 ) and antisense oligomer, nt 1376 to 1394 (50 -GCATTGTATGTGACACGCC-30 ), pair 3 sense oligomer, nt 1139 to 1158 (50 -ATTGACCTGCTGGACACACT-30 ) and antisense oligomer, nt 2457 to 2474 (50 -TGGAAAGCAGGTGTGAGA-30 ), pair 4 sense oligomer, nt 2165 to 2184 (50 -ACCAGCCGGGCTGTGCTGGT-30 ) and antisense oligomer, nt 3651 to 3672 (50 -ACCTGGCTGCGCGGAACTGCAT-30 ), pair 5 sense oligomer, nt 3224 to 3242

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(50 -CTCTTGGCTGAGGTCAAGGA-30 ) and antisense oligomer, nt 4222 to 4242 (50 -GCCCACTTGACTTAGTTCTTG-30 ). Double-stranded DNA sequencing was performed by GenScript, Inc. (NanJing, China) and the data was deposited in GenBank (Accession number JN983806).

Construction of the full-length ROND160E2E3 cDNA and its expression in HEK293 cells The 1.3 and 1.0 kb PCR fragments covering the nucleotide sequence from 1139 nt to 2474 nt were amplified from the RT products using oligomer pair 3 (1139 nt to 1158 nt and 2457 nt to 2474 nt). The fragments were then subcloned into the pGEM-T Easy vector (Promega). After confirming the deleted regions in the 1.0 kb fragment by DNA sequencing, the cDNA fragment was digested with the restriction enzymes Eco47III and NheI. The resulting 700 bp fragment, designated DRON cDNA, was isolated. This fragment is shorter than the regular wtRON PCR product and has an in-frame deletion of 318 nt. To construct the ROND160E2E3 cDNA, the vector pGEM-T Easy (Promega) containing a full-size wild-type RON cDNA (pGEM-T wtRON) was digested with Eco47III and NheI to eliminate a 1 kb fragment. The digested pGEM-T wtRON fragment was then ligated with the 0.7 kb DRON cDNA fragment to create the full-length ROND160E2E3 cDNA (pGEM-T DRON). Then the mammalian expression vector pcDNA4 HisMaxC (Invitrogen) containing a full-size wild-type RON cDNA (pcDNA4 HisMaxC wtRON) was digested with NotI to eliminate a 4.2 kb fragment. The digested pcDNA4 HisMaxC wtRON fragment was then ligated with the 3.9 kb DRON cDNA fragment to create the full-length ROND160E2E3 cDNA (pcDNA4 HisMaxC DRON). Transfection of HEK293 cells with the pcDNA4 HisMaxC-DRON vector was performed using the FuGENE-HD (Roche) transfection reagent. Cells were selected for 2 weeks using 100 mg/ml Zeocin. Individual cells were cloned and analyzed for the expression of ROND160E2E3 by Western blotting with rabbit IgG to RON peptide. Cells expressing ROND160E2E3 were pooled and used in the indicated experiments. Control clones were obtained by transfecting empty vector.

Cell surface biotinylation and immunofluorescent staining Cell surface biotinylation was performed using a Cell Surface Protein Isolation Kit (Thermo Fisher Scientific, Rockford, IL, USA) as instructed by the supplier. Cells were first labeled with EZLink Sulfo-NHS-SS-Biotin, a thiol-cleavable amine-reactive biotinylation reagent. Cells were subsequently lysed with a mild detergent, and the labeled proteins were then isolated with NeutrAvidin Agarose. The bound proteins were released by incubating with SDS-PAGE sample buffer containing 50 mM DTT. Proteins were subjected to SDS-PAGE and Western blotting with H-160 antibody. The immunofluorescent staining was performed as described previously [23]. Primary antibodies against the RON a-chain was added followed by secondary antibodies conjugated with FITC. Cells incubated with normal mouse IgG were used as controls. Sections were counterstained with DAPI. All slides were examined and photographed under Olympus BX51 upright microscope equipped with fluorescent devices.

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Cell transwell migration and wound healing assay The cell transwell migration assay was performed by using Polyester Membrane 24 Well Transwell (8 mm, Corning, NY, USA) filters. The lower chambers of the migration filters were filled in duplicate with 600 ml of DMEM containing different amounts of MSP. Cells were plated in a volume of 100 ml serumfree DMEM per Transwell filter at a density of 1  106. Cells were allowed to migrate in 5% CO2 at 37 1C for 6 h and were subsequently fixed by immersion of the filters in methanol at room temperature for 15 min. Filters were washed with deionized water and stained in 0.2% crystal violet in a 20% methanol/water solution for 10 min. The cells were removed from the upper surface of the membrane with a cotton swab. The cells that had migrated to the underside of the membrane were counted at 200  magnification from five random fields on each membrane. Wound healing assays were used to determine the ability of cells to migrate to cover the open space. Cells were grown to confluence and mechanically scratched to remove a fixed area of cells. Cells were stimulated with or without MSP (2 nM) for 24 h, and then photographed for analysis.

Statistics All experiments were repeated at least three times and the results were expressed as mean7SE. All statistical analyses were performed using Student’s paired or unpaired t-tests. Po0.05 was considered significant.

Results Detection of a 160 kDa RON isoform in the Human colon carcinoma cell line HCT116. After immunoprecipitation with a carboxy-terminus-specific antibody against the RON receptor (C-20), cell lysate from HCT116 and Colo320 cells was subjected to Western blotting with a different C-terminus antibodies against the RON receptor [23]. This identified a distinctive band with a molecular mass of 160 kDa (Fig. 1A), which represents a short form of the RON protein that is expressed in the HCT116 cell line. A RON variant with a similar molecular weight, ROND160, had been previously identified in the human colon carcinoma cell line HT29 as an autophosphorylated oncogenic variant [19]. It was determined by Western blotting that ROND160 was composed of a 160 kDa band of pro-RON and a 125 kDa band of a b chain. Because ROND160 was a product of the deletion of exon 5 and exon 6, which encode the amino acids from 574 to 683 of the Ron protein, we used a specific antibody against amino acids 531–690 of the Ron receptor (H-160), which cannot detect the ROND160 protein, for Western blotting of HCT116 lysate. A band with a molecular mass of 160 kDa was also present (Fig. 1B), which indicated that a potentially novel RON variant. But no band can be detected in Colo320 cell line with the two antibodies (Fig. 1A and B). Eight colon epithelial cell lines were then used to determine the expression of RON expression using H-160 antibody. The results are shown in Fig. 1C. The 160 kDa band can be detected in HCT116, RKO, SW620 and SW480 cells. But it was barely detectable in DLD1, HCe8693, HT29 and colo320 cells.

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Fig. 1 – Expression of the RON isoform in human colorectal carcinoma cells. Cellular proteins from HCT116 and Colo320 colorectal tumor cell lines were immunoprecipitated with antibodies directed against the C-terminus of the RON b chain and separated by SDS-PAGE under reducing conditions. Rabbit IgG antibodies to the RON C-terminal peptide (A) or the extracellular domain of RON (H-160) (B) were used in Western blots. Blots were developed with ECL reactions. In the HCT116 cell line, both of the two RON antibodies identified a protein with an apparent molecular mass of approximately 160 kDa in addition to the 180-kDa pro-wt-RON. (C and D) Detection of RON and its variants in colorectal carcinoma cells (C) and human colorectal adenocarcinoma tissues (D). Immunoprecipitation was performed using cell lysates with a RON antibody (C-20) bound to protein A/G agarose beads. Proteins were separated in 7.5% SDS-PAGE under reduced conditions and transferred to the membrane. Western blotting was performed using Rabbit IgG antibodies to the extracellular domain of RON (H-160). HEK293 cells expressing wtRON were used as a wtRON control.

To determine if this novel RON variant is generated in primary colorectal adenocarcinomas, tissue lysates prepared from eight frozen tumor samples were analyzed by immunoprecipitation and Western blotting. The results are shown in Fig. 1D. The 160 kDa band can be detected in four of the eight tumor samples.

of RON variants, which depends on the molecular weight of protein. Thus, we named it ROND160E2E3 to reflect the deletion of exon 2 and exon 3. Fig. 2C shows the cDNA sequences obtained from these two fragments. The location of the deleted nucleotides and their corresponding amino acids in the 1.0 kb fragment are also indicated.

Molecular cloning of the RON variant The full-length cDNA was cloned from HCT116 cells to investigate whether exons were deleted in the cDNA encoding for the novel RON protein. We designed five pairs of oligonucleotide primers according to the previously reported RON sequence [7] to perform RT-PCR. The five amplified overlapping segments covered the entire RON cDNA from position 183 to position 4242 (Fig. 2A). Two distinct fragments were identified through amplification of the region between 1139 nt to 2474 nt: the normal fragment (1.3 kb) and a smaller fragment (1.0 kb). As the molecular weight of the new RON protein is 160 kDa, we hypothesized that the 1.0 kb band may represent the new protein. After isolating and subcloning both fragments into the pGEM-T vector, both fragments were sequenced. It is clear that the smaller fragment lacked 318 nt between positions 1259 nt to 1576 nt; this results in an in-frame deletion of 106 amino acids in the b chain. The absent section is the exact site of exon 2 and exon 3 (Fig. 2B). As the molecular weight of this novel variant is similar to ROND160, an exon 5 and exon 6 deletion variant, the novel RON variant cannot named by the customary naming rule

Transfection and expression of ROND160E2E3 in HEK293 cells Two PCR products, 1.3 kb and 1.0 kb, were identified when the region between 1139 nt and 2474 nt was amplified. The product of the 1.0 kb fragment, created by the deletion of exon 2 and exon 3, was subcloned into the pGEM-T Easy vector. In the pGEM-T Easy vector containing the full length cDNA of wtRON, the Eco47III-NheI fragment was replaced with the same restriction enzyme digested product containing the exon 2 and exon 3 deleted fragment. Thus, we obtained the full length cDNA of ROND160E2E3 in pGEM-T Easy. The full length cDNA of ROND160E2E3 was then ligated into the pcDNA4 HisMaxC mammalian expression vector and transfected into HEK293 cells by FuGENE-HD system according to the manufacturer’s instructions. Transfected cells were confirmed by Western blotting with H-160 and C-terminus antibodies against the RON receptor (Fig. 3A and B). Under reducing conditions, HEK293-wtRON cells had two bands, the 180 kDa pro-RON band and the 150 kDa Ron b chain band. In contrast, HEK293-ROND160E2E3 cells had only

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Fig. 2 – D-RON protein originates from an alternatively spliced mRNA. (A) RT-PCR was performed to amplify partially overlapping sequences of the entire RON cDNA. The PCR amplification fragments were resolved by electrophoresis in 1% agarose gel. Two distinct fragments were identified when the region between 1139 nt to 2474 nt (primer pair 3) was amplified. The difference in the size of the cDNA clones corresponded to the length of the deletion (approximately 300 bp). (B) PCR amplification product from primer pair 3 was sequenced. According to the wtRON cDNA, there is a deletion of 318 bp from the end of exon 1 to the beginning of exon 4, which indicated the deletion of exon 2 and exon 3. (C) Partial nucleotide sequence of the RON cDNA fragment obtained from HCT116 cells. RT-PCR and DNA sequencing were performed as described in section ‘‘Materials and methods’’. The deleted 106 amino acids and corresponding 318 nucleotides in ROND160E2E3 are underlined with a solid line. The cysteine residues in the sequences are boxed.

one 160 kDa band, confirming that the ROND160E2E3 protein is a single-chain protein with an uncleaved b chain.

ROND160E2E3 lacks tyrosine phosphorylation, but Akt was activated in ROND160E2E3 expressing cells The tyrosine phosphorylation status of ROND160E2E3 was studied in HEK293 cells. HEK293-wtRON cells were used as a positive control. After immunoprecipitation with C-20, Western blotting with p-Tyr-100 showed that the ROND160E2E3 protein was not phosphorylated on tyrosine whether the cells had been stimulated with MSP or not (Fig. 3C). In contrast, tyrosine phosphorylation of the wtRON b chain was observed after MSP stimulation as described previously [10]. Protein loading was comparable as shown by re-probing of the blot with an anti-RON antibody. We then measured the kinase activity of wtRON and ROND160E2E3. The HEK293-wtRON cells showed low level of kinase activity

without MSP stimulation. After stimulation with 2 nM MSP, high levels of kinase activity were observed in HEK293-wtRON cells. But the kinase activity in HEK293-ROND160E2E3 cells was as low as in HEK293 vector control cells, whether in the presence of 2 nM MSP or not (Fig. 3D). These results, together with data shown in Fig. 3C, suggest that ROND160E2E3 lacks tyrosine kinase activity in HEK293-ROND160E2E3 cells. The MAP kinase pathway and phosphatidylinositol (PI)-3 kinase pathway [23,25] are two pathways that are usually downstream of the RON receptor tyrosine kinase. Phosphorylation of RON Ser-1394 has been associated with the PI-3K/Akt pathway and can provide a docking site for 14-3-3 proteins to facilitate cell movement [24]. Western blotting was used to detect ERK1/2 and Akt phosphorylation to evaluate the activity of the MAP kinase and PI-3 kinase in HEK293 cells transfected wtRON or ROND160E2E3 (Fig. 4). ERK1/2 phosphorylation was barely detected in HEK293-wtRON cells in the absence of MSP.

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Fig. 3 – Expression and tyrosine phosphorylation ability of ROND160E2E3 in HEK293 cells. HEK293 cells were transfected with wtRON or D-RON cDNA, and the proteins were analyzed as described. RON antibodies against the C-terminal (A) and extracellular domain (H160) (B) were used to detect RON expression. The new variant is confirmed to be a single-chain protein. (C) Cells (3  106/ml) were stimulated with or without 2 nM MSP at 37 1C for 10 min. Immunoprecipitation was performed using cell lysates with a RON antibody (C-20) bound to protein A/G agarose beads. Proteins were separated by 7.5% SDS-PAGE under reducing conditions and transferred to a membrane. The phosphorylated tyrosine of RON was detected by anti-p-tyr-100 antibodies. The membrane was treated with stripping buffer and re-probed with antibodies to the extracellular domain of RON (H160) to detect the expression of RON proteins. GAPDH was used as the loading control. (D) In vitro tyrosine kinase assay in HEK293-wtRON and HEK293-ROND160E2E3 cells. Cells were serum-starved overnight and pretreated with 2 nM MSP for 5 min before lysis. The HEK293 cells expressing an empty vector were set as negative control. The in vitro kinase assay was performed as detailed under section ‘‘Materials and methods’’. Results are expressed as means7SD. (Po0.05, compared to wtRON MSPþ). After stimulation with 2 nM MSP, ERK1/2 phosphorylation was dramatically increased. In contrast, ERK1/2 phosphorylation could not be detected in HEK293-ROND160E2E3 cells either in the absence or in the presence of MSP, which indicated that ROND160E2E3 expression cannot activate the MAP kinase pathway. Akt phosphorylation was detected at low levels in HEK293wtRON cells in the absence of MSP and increased approximately fourfold in the presence of 2 nM MSP. In HEK293-ROND160E2E3 cells, Akt was moderately phosphorylated either in the absence or in the presence of 2 nM MSP, this phosphorylation level was approximately twofold compared to the level Akt phosphorylation in HEK293-wtRON cells in the absence of MSP.

ROND160E2E3 protein expressed in the intracellular compartment Of the known RON variants, ROND160 and ROND170 have the mature RON b chain expressed on the cell membrane [19,22]. In

contrast, ROND165 and ROND155 have a single-chain form and are present in the cytoplasm with no mature b chain [17,20]. The single-chain form of ROND165 and ROND155 is expressed in the cytoplasm and cannot be further activated by MSP stimulation [17,20]. The single band result of immunoprecipitation and Western blotting with a RON specific antibody indicated that the ROND160E2E3 variant was a single-chain form RON variant. As a rule, only proteins that have attained the proper threedimensional structure are secreted or expressed on the cell surface. Folding and assembly intermediates are retained intracellularly [17]. Thus, we hypothesized that the ROND160E2E3 protein may localize in the intracellular compartment. To confirm our hypothesis, a cell surface labeling experiment was conducted in HEK293-wtRON cells and HEK293-ROND160E2E3 cells to determine if the ROND160E2E3 proteins are on the cell surface or in the cytoplasm. Cells were first labeled with EZ-Link Sulfo-NHS-SS-Biotin, then were subsequently lysed with a mild detergent and the labeled proteins were then isolated with

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NeutrAvidin Agarose. The flow-through fraction contained the cytoplasmic proteins, and the eluent from agarose contained the cell surface proteins. The two cell lysates were subjected to

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SDS-PAGE and Western blotting using the H-160 antibody. The result showed that the wild-type RON protein was only expressed on the membrane in two forms, whereas the ROND160E2E3 protein was mostly expressed in cytoplasm in a single band (Fig. 5A). Immunofluorescent staining using antibodies against the RON a-chain confirmed the result that ROND160E2E3 protein was localized in the cytoplasm (Fig. 5B).

ROND160E2E3 induces cell motility through PI-3K pathway

Fig. 4 – Constitutive activation of the PI-3K/Akt pathway in HEK293-ROND160E2E3 cells. HEK293-wtRON or HEK293ROND160E2E3 cells (2  106/ml) were serum-starved overnight and then stimulated with or without 2 nM MSP at 37 1C for 10 min. After treatment, cellular proteins (50 lg/sample) were separated by 8% SDS-PAGE and blotted with antibodies to phosphorylated p42/44, pan p42/44, phospho-Akt (Thr308) and pan-Akt. RON expression was also detected with anti-RON antibody (H-160). GAPDH was used as the loading control.

HEK293-wtRON cells migrated in a MSP concentration-dependent manner (Fig. 6A). HEK293-ROND160E2E3 cells migrated both in the absence of MSP and in varying concentrations of MSP. The number of migrated HEK293-wtRON cells paralleled the number of HEK293wtRON cell that migrated in the presence of 0.5 nM MSP. As a vector control, HEK293 cells transfected with the empty vector barely migrated even with MSP stimulation. Migration of HEK293ROND160E2E3 cells was also determined by measuring cells that moved into the open space on the surface of a culture dish. As shown in Fig. 6B, expression of wtRON moderately increases spontaneous migration of 293 cells. More than 80% of the open space was covered within 24 h by migrated cells. This effect was further enhanced when MSP was used. While ROND160E2E3 expression significantly increases migration, almost 100% of the open space was covered within 24 h by migrated cells with or without 2 nM MSP stimulation. As mentioned above, the ROND160E2E3 protein does not have tyrosine phosphorylation but can activate the PI-3K/Akt pathway. The PI-3K/Akt pathway activation was previously reported to promote cell migration [24]. We thus used a PI-3K/Akt pathway inhibitor, LY294002, to see if the enhanced migration ability of HEK293-ROND160E2E3 cells is dependent on the activation of the PI-3K/Akt pathway. After treating with 10 mM

Fig. 5 – ROND160E2E3 protein expressed in the intracellular compartment. (A) HEK293-wtRON and HEK293-ROND160E2E3 cells were subjected to cell surface biotinylation. The flow-through fraction (cytoplasmic proteins) and cell surface proteins eluent from agarose were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose. The RON antibody (H160) was used to detect RON expression. The 180-kDa pro-RON and mature 150-kDa RON protein were expressed on the cell surface and the ROND160E2E3 protein was retained in the intracellular compartment. C: cytoplasm; M: cell membrane. (B) Detection of RON expression by immunofluorescence staining on HEK293-wtRON and HEK293-ROND160E2E3 cells. Note: wtRON is localized to the cell surface in HEK293-wtRON cells, while ROND160E2E3 is diffusely expressed in cytoplasma of HEK293-ROND160E2E3 cells. Nuclei were counter-stained with DAPI (blue). (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6 – ROND160E2E3 induces a motile phenotype in vitro. (A) The effect of ROND160E2E3 on cell migration. A transwell migration assay was performed to measure the migratory ability of HEK293 cells expressing RON and ROND160E2E3. As a control, HEK293 cells expressing an empty vector were also studied. The assay was performed as described in section ‘‘Materials and methods’’. Results are expressed as means7SD. (B) Wound healing assay for HEK293-ROND160E2E3 cells. Experiments were performed as described above. After stimulated with or without 2 nM of MSP for 24 h, cells were photographed.

LY294002, the HEK293 cells expressing either wtRON, ROND160E2E3 or the control vector were subjected to the migration assay. Results showed that the migratory ability of HEK293ROND160E2E3 cells treated with LY294002 was reduced markedly and was similar to HEK293 vector control cells. The MAPK inhibitor PD98059 did not affect the migratory ability of HEK293-ROND160E2E3 cells. In contrast, LY294002 or PD98059 alone had only a slight effect on the migratory ability of HEK293wtRON cells. Combination of the two inhibitors significantly inhibited the cells’ movement (Fig. 7A). Similar results were obtained in wound healing assay (Fig. 7B). These results indicated that ROND160E2E3 promotes cellular migratory ability through the PI-3K pathway.

Discussion We identified and cloned a novel splice variant of RON from the human colorectal cancer cell line HCT116. This variant has no tyrosine phosphorylation, but transfected HEK293 cells showed

an increased migratory activity. As the molecular weight of the novel variant was 160 kDa, similar to ROND160, the novel RON variant cannot simply named by the customary naming rule of RON variants, which depends on the molecular weight of protein. Thus, we named it ROND160E2E3 to reflect its molecular weight and the deletion of exon 2 and exon 3. ROND160E2E3 differs from the wild-type RON by lacking 106 amino acids in the extracellular domain. As described in the molecular cloning experiment, two PCR products of 1.3 kb and 1.0 kb were found when the region between 1139 nt to 2474 nt from HCT116 cells was amplified. DNA sequencing confirmed that a deletion of 318 nt occurred in the 1.0 kb fragment. Analysis revealed an in-frame deletion of 106 amino acids in the extracellular domain, which corresponded to a deletion of exons 2 and 3. The deletion may induce an imbalance of cysteine residues and thus conceals the exposure site for proteolytic enzymes, which results in the natural accumulation of a single chain pro-ROND160E2E3 formed in the cytoplasm. Four types of RON activation have been discovered including activation by MSP stimulation, over-expression of the RON

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Fig. 7 – ROND160E2E3 induces cell migration through PI-3K pathway. (A) Inhibition of the PI-3K/Akt pathway inhibited ROND160E2E3 induced cell migration ability in HEK293 cells. The migration assay was performed on HEK293 cells expressing RON and ROND160E2E3 either in the absence or in the presence of 2 nM MSP, 10 lM LY294002 or 50 lM PD98059. The assay was performed as described. Results are expressed as the means7SD. (Po0.05, compared to ROND160E2E3 treated with LY294002 or LYþPD). (B) Cells were grown to confluence and mechanically scratched to remove a fixed area of cells. Cells were stimulated with MSP (2 nM), and chemical inhibitors such as LY294002 (10 lM) or PD98059 (50 lM) were added simultaneously. Photographs were taken after 24 h. CTRL: vector control.

receptor, formation of a RON splice variant and mutation in the kinase receptor domain [15,26]. Splice variants play an important role in RON autoactivation [17,19,20], which indicates that RON splice variants may have an important role in tumor invasiveness and metastasis in colorectal cancer. We found that the tyrosine domain of ROND160E2E3 cannot be phosphorylated spontaneously or by stimulation with MSP. HEK293ROND160E2E3 cells migrated stably both in the absence of MSP and in varying concentrations of MSP. The number of migrated HEK293ROND160E2E3 cells paralleled the number of HEK293-wtRON cells when stimulated with 0.5 nM MSP. Enhanced migratory ability

indicates that a signaling pathway downstream of ROND160E2E3 might be activated. Two common downstream pathways, p-ERK1/2 and p-Akt, were examined in HEK 293-ROND160E2E3 cells. Constitutively active p-Akt was detected, but p-ERK1/2 was not activated either with or without MSP stimulation. The PI-3K pathway is very important in the RON signaling pathway, which participates in the epithelial cell migration induced by MSP stimulation; additionally, the activation of PI-3K depends on the stimulation of MSP [25]. As observed in our study, p-Akt activation in 293-ROND160E2E3 cells was spontaneous and did not depend on MSP stimulation. Several proteins were reported to regulate Akt activity, which including

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PTEN (phosphatase and tensin homolog deleted on chromosome 10) [27], PP2A (protein phosphatase 2A) [28], and PHLPP (pleckstrin homology domain leucine-rich repeat protein phosphatase) [29]. The mechanism of Akt activation in this study is still unclear. Six RON splicing variants, including ROND165, ROND160, ROND155, ROND55, ROND170, and ROND90, have been previously identified [13,17–19,21,22]. Four variants, ROND165, ROND160, ROND155 and ROND55, are expressed in colorectal cancer cell lines and can increase the motility and invasive capabilities of cancer cells [17,19–21]. We report here a novel variant of RON, ROND160E2E3, which was also found in a colon cancer cell line. The molecular weight of this variant is 160 kDa, which is similar in molecular weight to the ROND160 variant. Under non-reducing conditions, they both show a single 160 kDa band by Western blotting. In contrast, under reducing conditions, the ROND160 variant had two distinctive bands, while the ROND160E2E3 variant only had one band. This is similar to ROND165, which was the first variant of RON discovered in the gastric cancer cell line KATOIII [17]. The ROND165 variant lacks the 49 amino acids encoded by exon 11. Among the missing 49 amino acids are three cysteine residues normally involved in intramolecular disulfide bonds. Therefore, the imbalance of the cysteine pairs renders the uneven cysteines available for intermolecular disulfide bonding with other D-Ron partners, which creates oligomers. There is one cysteine in the missing 106 amino acids of ROND160E2E3 protein. The lost cysteine may cause an imbalance of the cysteine pairs and create oligomers. Thus, the ROND160E2E3 protein cannot attain the proper three-dimensional structure and is retained intracellularly. Among the 6 previously described variants, ROND170, ROND165, ROND160, ROND155 and ROND55 have the tyrosine region. ROND170 doesnot have tyrosine kinase activity [22]. This characteristic is similar to ROND160E2E3. However, ROND170 is a dominant-negative variant that has the potential to inhibit RONmediated tumorigenic activities in colorectal cancer cells [22]. Although ROND160E2E3 does not have tyrosine phosphorylation ability, this variant has constitutive activation of the Akt pathway. The activation of Akt pathway promotes epithelial cell migration, which is different from ROND170. While the lack of phosphorylation in the tyrosine kinase region is different from the tumorigenic variants ROND165, ROND160, ROND155 and ROND55, we propose that the novel variant is a distinct variant that can promote cell migration without tyrosine phosphorylation. This suggests that there may be other pathways that can induce motile ability in RTKs.

Conclusions Our data describes a new splice variant of RON and suggests a novel role for the RON receptor in the progression of metastasis in colorectal cancer. The molecular weight of this variant is 160 kDa and we named it ROND160E2E3. This variant is a singlechain protein and expressed in the intracellular compartment lacks tyrosine phosphorylation ability, but it has constitutively activated Akt activity in transfected HEK293 epithelial cells. The expression of this variant in HEK293 cells resulted in an increased migratory activity in vitro mediated through the PI-3K/Akt pathway.

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Acknowledgments This work was supported by grant from the National Natural Science Foundation of China (NSFC-81071962) and Program for Innovative Research Team in Zhejiang Province (Grant no. 2010R50046).

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