The findings in this study demonstrate that alterations in the first IPT unit in the RON extracellular sequence results in two novel variants with different biological profiles. Structurally, the IPT units consist of 80 to 100 amino acids and are featured by immunoglobulin-like fold [
25,
43]. The IPT units are also found in certain transcription factors such as NF-κB and c-Jun, where it is involved in protein-protein and/or protein-DNA interaction [
44]. The significance of the IPT units in MET and RON has recently been discovered and emphasized. The deletion of the first IPT unit in the RON extracellular sequences converts wild-type RON into oncogenic agent RON160 [
13], although the underlying mechanisms are unknown. In MET, the fourth IPT unit in the β-chain extracellular sequence harbors a high affinity binding site for ligand HGF/SF [
45]. HGF/SF binding to this IPT unit is essential for induction of MET activation [
45]. Clearly, these findings illustrate the importance of the IPT units in MET/RON-mediated signaling cascades and tumorigenic activities. The data from our current studies demonstrate that deletion or insertion in the RON first IPT unit exerts different consequences. Although deletion of the first IPT unit leads to oncogenic conversion, insertion of 20 amino acids in the same unit is not sufficient to transform the RON protein into an oncogenic agent. However, insertion has important impact on biochemical properties of RON. We show that proteolytic conversion of pro-RON
E5/6in into the two-chain mature protein by convertase furin is significantly delayed upon precursor synthesis. RON
E5/6in is also highly susceptible to cell-associated serine proteases, which act on a short sequence leading to generation of another variant RONp110. Moreover, RON
E5/6in is internalized in an accelerated manner upon anti-RON mAb engagement. Thus, alterations in the first IPT unit differentially regulate RON-mediated activity with different biochemical properties. In addition, generation of RON160 and RON
E5/6in provides an opportunity to understand the roles of IPT units in regulating RON activation and activity, which could aid to develop therapeutic agents for inhibition of RON-mediated tumorigenic signaling.
Overexpression of RON in cancerous tissues is often accompanied with the generation of aberrant mRNA transcripts and their corresponding variants [
13,
14]. This has been considered as a mechanism by which RON displays its protein diversity and regulates epithelial homeostasis and malignant transformation [
7]. A survey by PCR of primary colon, lung, breast, and brain tumor samples has revealed that aberrant mRNA transcripts encoding for known and unknown variants such as RON165, RON160, and RON155 were wildly produced with relatively high frequencies in colon, breast, lung and other types of cancers [
46]. These variants are mainly generated by aberrant mRNA splicing processes that delete exon 11 (RON165), exons 5 and 6 (RON160), and exons 5, 6 and 11 (RON155) [
7,
46]. It needs to be emphasized that exon 11 encodes 49 amino acids belonging to the fourth IPT unit in the RON β-chain extracellular sequences [
25], which is required for pro-RON maturation and cell surface localization [
28]. Results in current studies demonstrate that alterations in the first IPT unit in the RON protein are not a rare occurrence. Among 12 cancer cell lines analyzed, abnormality in the first IPT unit was observed in 5 cell lines originating from colon, breast and pancreatic tumors. These results are consistent with those from analysis of primary tumor samples [
14,
46]. As reported, deletion of exons 5 and 6 were observed in more than 50% of primary colon and 90% of brain tumor samples but not in any normal tissues [
14,
46]. Further analysis of insertions between exons 5 and 6 using clinical tumor samples would be very informative. Although the underlying mechanisms of variant generation are currently unknown, it is known that aberrant splicing and intron retention in receptor tyrosine kinases occur commonly in cancer cells [
47,
48]. Considering the oncogenicity is of RON160
in vivo, such alterations with high frequencies should have pathogenic significance in relevance to tumor progression and malignant phenotypes.
From the viewpoint of disrupting the first IPT unit, it was a surprise that RON
E5/6in differs significantly from RON160 in terms of their biochemical and biological properties (Table
2). First, RON160 and RON
E5/6in both are cleaved from precursor into respective mature forms but the kinetics of their processing is different (Figure
4). Pro-RON160 is cleaved at a rate similar to that of pro-RON. In contrast, RON
E5/6in is matured at relatively late stages. This is probably due to relative insensitivity of RON
E5/6in to convertase furin-mediated proteolytic cleavage. Such insensitivity could be due to sequence alterations in the insertion. Site-directed mutagenesis may verify if this is the case. Another possibility is that insertion-induced conformational change may affect the access of furin to the cleavage site located at α/β chain junction. Second, under regular culture conditions containing FBS, RON
E5/6in is the major source for generation of RONp110, although wild-type RON can also be truncated by exogenous trypsin to form RONp110. This suggests that the insertion causes the digestion site (Val
629-Pro-Arg-Lys-Asp-Phe) in the first IPT unit more accessible to cell-associated trypsin-like serine proteases. As a post-translational truncated product, RONp110 misses the majority of the extracellular domains including sema, PSI, and a large portion of the first IPT. MSP stimulation hardly induced its phosphorylation (Figure
1B and
2C), which suggests that MSP may not bind to RONp110. As expected, enzymatic digestion of RON or RON
E56in by cell-derived trypsin-like proteases also produce a soluble ~80 kDa RON extracellular isoform (RON
Er80) comprising the entire 35 kDa α-chain and a ~45 kDa partial extracellular β-chain. The isoform is similar to a previously reported RONΔ85 [
24]. RONΔ85 is a soluble truncated RON variant produced by a mRNA transcript from a breast cancer cell line with insertion of 49 nucleotides between exons 5 and 6 [
24]. RONΔ85 has the inhibitory effect on MSP-induced RON signaling events [
24]. Considering their structural similarities, it is reasoned that the RON
Er80 may have the ability to regulate MSP-induced RON-mediated activities. Currently, the role of RONp110 is unknown. Interestingly, a similar variant of MET lacking the ectodomain but retaining the transmembrane and intracellular domains has been discovered in several cancer samples [
49]. This protein resides on the cell surface and displays transforming, invasive, and tumorigenic activities [
49]. Third, deletion of the first IPT unit results in constitutive tyrosine phosphorylation [
13]. In contrast, insertion does not convert RON
E5/6in into constitutive phosphorylation. RON
E5/6in remains inactive and requires MSP stimulation for phosphorylation and activation of downstream signaling molecules such as Erk1/2 and AKT (Figure
2C). Previous studies have shown that deletion of first IPT unit results in imbalance of cysteine residues in the extracellular sequences, a possible reason for spontaneous dimerization leading to constitutive phosphorylation [
13]. Interestingly, a cysteine residue was seen in the inserted 20 amino acids in the RON
E5/6in molecule, which also causes an imbalance in the number of cysteine residues in the extracellular sequence. However, such addition does not seem to affect the extracellular conformation of RON
E5/6in. Thus, additional mechanism(s) is probably involved in constitutive activation of RON160. Fourth, RON160 is relatively resistant to anti-RON mAb-induced internalization and degradation. In contrast, RON
E5/6in is highly susceptible to Zt/g4-mediated degradation (Figure
5). At present, we do not know mechanism(s) responsible for such an accelerated process. However, this is important for RON160 to sustain its intracellular oncogenic signaling. As reported previously, oncogenic RON variants created by mutations in the kinase domain are highly resistant to ligand-induced internalization [
50]. We have previously found that anti-RON mAb-induced down-regulation attenuates RON-mediated tumorigenic signaling and motile-invasive activities in colon cancer cells [
39]. Thus, insertion in the first IPT unit, through an unknown mechanism, accelerates antibody-induced RON
E5/6in internalization and degradation. Finally, insertion and deletion in the first IPT showed differential effects on cellular activities. From functional analysis, RON
E5/6in mediated EMT-like activities are similar to those mediated by wild-type RON. However, as judged by levels of vimentin and E-cadherin, changes in cell morphologies, and cell motility, RON160 is much more potent than RON
E5/6in in mediating these tumorigenic activities. Analysis of cell transforming and anchorage-independent activities further demonstrate that insertion in the first IPT unit does not convert wild-type RON into a transforming agent. It is the deletion that renders RON160 as the transforming variant. As evident by
in vitro transforming assays, the number of foci mediated by RON160 was significantly higher than that in RON
E5/6in expressed NIH3T3 cells. Anchorage-independent growth by colonies in soft agar was also observed only in RON160 expressing NIH3T3 cells. Thus, alterations in the first IPT unit, either by insertion or deletion, result in two RON variants with distinct structural and cellular activities.
Table 2
Biochemical and Biological Differences between RON160 and RONE5/6in
mRNA in tumor cell lines | 11 out of 12 in CC, BC, PC lines | 4 out of 12 in CC, BC lines | 4 out of 12 in CC, BC, PC lines. |
First IPT unit | wild-type | exon deletion | intron retention |
Protein location | cell surface | cell surface | cell surface |
Activation | MSP required | constitutively active | MSP required |
Response to MSP | strong | moderate | strong |
Trypsin digestion | sensitive | no effect | sensitive |
Generation of RONp110 | upon trypsin treatment | no effect | spontaneous and trypsin treatment |
Furin treatment | sensitive | sensitive | less sensitive |
Intracellular degradation | sensitive | less-sensitive | highly sensitive |
Induction of EMT | moderate effect | strong effect | moderate effect |
Cell migration | moderate effect | highly effect | moderate effect |
Transforming activity | no effect | strong effect | no effect |
Colony formation | no effect | strong effect | no effect |