{'fraction_non_alphanumeric': 0.028614940686117345, 'fraction_numerical': 0.027893555626803464, 'mean_word_length': 5.222942643391521, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
RON kinase isoforms demonstrate variable cell motility in normal cells

Introduction Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Methods Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. Results RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). Conclusions RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.


Introduction
Aberrant RON (Recepteur d' Origine Nantais) is a receptor tyrosine kinase member of the MET proto-oncogene family with known involvement in both tumor progression and metastasis [1,2,3,4,5,6,7]. In normal cells, RON is transiently activated through binding with its ligand, macrophage-stimulating protein (MSP), which initiates receptor autophosphorylation and upregulation of kinase activity [8,9,10]. However, RON is likely abnormally activated through overexpression of receptor proteins or point mutations in the kinase domain, leading to dimerization [11]. RON is not only directly implicated in tumorigenesis and invasiveness, but also interacts downstream with oncogenic pathways phosphatidyl inositol-3 kinase (PI3 K)/Akt and Ras/Raf/mitogen-activated protein kinase (MAPK) [12,13,14]. RON also participates in cross-talk with other oncogenic receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR) and Met receptor [3,7]. Constitutive activation of the RON receptor causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis [12,15]. Both in vitro and in vivo experiments have shown RON to be capable of inducing invasive phenotypes and distant tumor metastasis [14,16,17].
RON receptor splice variants are increasingly implicated in mechanisms underlying oncogenesis, metastasis and the creation of drug-resistant phenotypes [18,19,20]. While generally the creation of isoforms is thought to positively contribute to protein diversity, normal expression of splice variants can be significantly altered in cancer [21,22]. In fact, variants are estimated to be abnormal in over half of cancers studied in the literature, particularly those with high cellular proliferation such as epithelial cancers [23]. Wildtype RON is a 180-kDa heterodimer containing 20 exons and 19 introns, composed of an extracelullar 40-kDa alpha chain and a 150-kDa transmembrane beta chain containing an intracellular kinase [6]. Deletion or truncation of RON receptor mRNA transcripts may result in splice variants that demonstrate constitutive autophosphorylation and increased kinase activity [24].
Three commonly identified splice variants 155, 160 and 165 are generated by various exon deletions in different regions of the B-chain of the RON receptor, resulting from abnormal mRNA splicing [20]. The major known characteristics of these isoforms are summarized in Table 1. Both RON155 and RON 160 induce tumor formation in vivo and exhibit cell migration abilities in a ligand-independent manner [20,26]. RON 165, discovered in 1996 in gastric carcinoma cell line KATO-III [24], is capable of inducing cell motility in transfected cells, though does not demonstrate transformative or tumorigenic potential.
RON plays a role in the metastasis of epithelial malignancies and presents an important potential therapeutic target for colorectal, ovarian, breast, gastric, lung and pancreatic cancer [19,27,28,29]. While advances in control of local disease have been made in recent years, breast cancer remains the second leading cause of death in women and colorectal cancer the third leading cause of cancer deaths in both sexes in the United States [30]. The sequelae of tumor metastasis accounts for the majority of cancer deaths. Approximately 20% of patients with colorectal cancer are found to have distant disease at the time of diagnosis [31]. While the development of monoclonal antibodies to wtRON is underway, little is known about the functional differences amongst RON isoforms RON155, RON160 and RON165, particularly differences in metastatic potential. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. We hypothesized that RON isoforms will demonstrate varying cell motility. (v/v) fetal bovine serum at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .

Plasmid transfection and immunoblotting analysis
Plasmid constructs for wtRON, RON155, RON160 and RON165 were transfected into HEK293 cells using Xfect Transfection Reagent (Clontech). We selected four individual clones for each isoform. The culture medium plus 800 μg/ml of G418 was used to select the clones.

Wound scratch assay
HEK293 cells, which were individually transfected with empty plasmid vector (pTagRFP as a control) or plasmids containing wtRON, RON155, RON160 and RON165 respectively were cultured in a 60-mm dish coated with collagen and grew until confluence. A uniform wound was created manually in the confluent monolayer of cells with a pipette tip. Wound closure was monitored at 0, 12 and 24 hours using a Nikon light microscope at 10x magnification. The percentage of wound coverage was measured using Image-J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA).

Statistical analysis
All experiments were repeated independently and in triplicate. Variance in cell motility values, if significant, was detected using first an F-test, followed by a

Low to undetectable levels of endogenous RON found in HEK293 cells
We found near undetectable endogenous RON expression in HEK cells compared to five commonly used colon cancer cell lines (Fig. 1a). This confirmed the utility of employing HEK293 cells for wtRON and RON isoform transfection and subsequent motility analysis.

WtRON and RON isoforms demonstrated increased cell motility compared to control cells
To determine differences in cell motility between RON and RON isoforms 155, 160 and 165 and control cells, a wound scratch assay was performed (Fig. 2). Migration was monitored at 12 and 24 hours. All RON cell lines were 100% confluent at 24 hours.  [ ( F i g . _ 2 ) T D $ F I G ]

RON 165 demonstrated increased cell motility compared to RON 160
The wound scratch assay analysis was also employed to compare differences between wtRON and the three RON isoforms. RON 165 was found to have significantly increased motility compared to RON 160 (37.37% vs. 32.40%; p = 0.03) (Fig. 2).

WtRON and RON isoforms have variable affects on PI3 K pathway downstream signaling
Relative quantity analysis demonstrated RON160 had increased levels of Akt, pGSK3β and FOXO3a protein expression compared to control, wtRON, RON 155 and165 (Fig. 4) all the cell lines studied. This is an interesting finding considering that wtRON has ligand-dependent activation while RON isoforms are constitutively activated. We suspect if MSP ligand was added there may be an even larger difference in cell motility. There continues a discussion as to whether wtRON is tumorigenic or promotes metastasis. Initial studies suggested that human wtRON was neither tumorigenic nor metastatic [4] However, further studies [11] showed wtRON to have transformative properties in NIH3T3 cells including long cytoplasmic extensions and loss of contact inhibition, even in the absence of ligand stimulation.
Zhou et al. confirmed wtRON to be incapable of tumorigenesis [20]. Eyob et al. also found knocking out RON had no effect on tumor development or growth rates, though eliminated lung metastasis [32]. Current knowledge suggests that while human wtRON may not have transformative properties, it is undoubtedly linked to the ability of tumors to metastasize.
Interestingly, RON 165 exhibited the fastest migration of the three RON isoforms. RON 165 is found in normal colorectal mucosa cells and is thought to contribute to metastatic spread and invasive phenotypes, though not tumorigenesis [20,24]. Conversely, RON160 is able to induce transformation, cell scattering and tumorigenesis in athymic nude mice. RON155, first reported by Zhou et al., was also found to induce cell scattering, transformation and tumor growth in vivo, though less efficiently than RON160. RON160 and 155 were found to cause multiple foci in transfected NIH3T3 cells, induce tumor formation and metastatic spread. However, in this study the metastatic potential of RON165 was not studied in vivo, as it was not tumorigenic in nude mice. Our study suggests that RON165 significantly increases cell motility. Overexpression of RON165 was noted in some types of breast and colon cancer, which may explain these cancers' extraordinary capacity for metastasis. Further experiments in an orthotopic model are needed to examine the true functional differences in metastatic potential of wtRON and RON isoforms, particularly RON165.
Understanding the ability of tumors to metastasize is of utmost clinical importance.
The invasion of metastatic disease to distant organs is the main cause of death in most cancer patients [33]. A hallmark of colorectal cancer is the ability to invade adjacent organs and metastasize to remote locations such as the liver or lung [34].
In normal colon epithelial cells, RON is barely expressed, though is highly expressed and constitutively activated in many colorectal cancer cell lines [33].
However, treatment of colorectal cancer is difficult as a significant degree of genetic heterogeneity leads to highly variable phenotypes [35]. The existence of splice variation adds yet another level of complexity to an individual patient's tumor. RON isoforms are related to deregulation of mRNA splicing machinery and not genetic mutations [15]. Research into splicing factors is currently developing, specifically examining the functions of a silencer and enhancer on exon 12 that may regulate aberrant splicing in epithelial cancers [18] or studying the SRSF2 Article No~e00153 gene which promotes inclusion of exon 11 [36,37] This study includes a few notable limitations. The three RON isoforms studied demonstrated increased cell motility compared to control cells as expected. In our study, RON 160 was increased compared to control, though did not reach statistical significance, which we attribute to a smaller sample size of available images. Our plasmid constructs were transfected into normal epithelial cells as opposed to a colon cancer cell lines. This is because many colon cancer cell lines have wtRON and RON splice variant expression [15,33]. This HEK293 cell model, having near undetectable levels of endogenous RON expression, allows for preliminary analysis of the impact of isolated splice variants on cell motility. Thus, we cannot yet comment on RON isoforms cell motility in tumor cells or how the differences in cell motility will translate in an in vivo animal model.
In this study we demonstrate that RON tyrosine kinase splice variants demonstrate variable cell motility in normal cells. Variation in cell motility between RON isoforms may represent a difference in metastatic potential. Future studies aimed at exploring the functional differences of RON isoforms in in vivo pre-clinical models will help to determine their role as potential therapeutic targets in both localized cancers and metastatic disease.