Splice variants of the extracellular region of RON receptor tyrosine kinase in lung cancer cell lines identified by PCR and sequencing

Abstract

Background: Altered expression of receptor tyrosine kinases (RTKs) is a major driver of growth and metastasis of cancers. Recepteur d'origine nantais (RON) receptor is a single-pass transmembrane RTK aberrantly expressed in a number of cancers. Efforts to block deregulated RON signaling in tumors using small molecule kinase inhibitors or antibodies are complicated by the presence of unknown number/types of isoforms of RON, which, despite having similar sequences, are localized differently and mediate varied functions. The objective of this study was to identify splice variants of RON transcripts between exons 1 and 10 that code for the extracellular region.

Methods: Direct cDNA sequencing was performed for the transcript between exons 1-10 of RON by Sanger sequencing in various lung cancer cell lines.

Results: PCR amplification and bi-directional sequencing of cDNA for section between exons 1 and 10 from lung cancer cell lines revealed the presence of several splice variants of RON transcripts; the variants were formed by skipping of exons 2, 2-3, 5-6, 6 and 8-9. Each of these transcript variants were found in one or more cell lines. While the variants formed by skipping of exons 2, 2-3 and 5-6 resulted in loss of 63, 106 and 109 amino acids, respectively, and didn't cause reading-frameshift, the transcripts formed by skipping of exons 6 and 8-9 caused reading-frameshift. Splice variant lacking exons 8-9 was found in 13 out of 23 cell lines tested.

Conclusion: Lung cancer cell lines contain several splice variants of RON which involve skipping of exons coding for extracellular region. Some of the splicing changes result in reading-frameshift and the N-terminally truncated isoforms are expected to be secreted out. The ubiquitous nature of alternative splicing events in RON suggests the need for isoform specific approaches to functional analysis and therapeutic targeting of RON.

Keywords: Alternative splicing; Lung cancer; Macrophage stimulating protein; RON; RON isoform; Receptor tyrosine kinase.

Fig. 1

Identification of splice variants of RON transcripts in lung cancer cell lines by PCR amplification and sequencing of cDNAs. a) RON splice variant lacking exons 2 and 3 in cell line H249. Sequencing from 5′ end showed an additional overlapping sequence starting at nucleotide 1231 of RON reference sequence and the overlapping sequence corresponded to 5′ starting sequence of exon 4. b) RON splice variants lacking exon 2 and exons 2–3 in cell line H82. c) RON splice variant lacking exons 5 and 6 in cell line H524. This indicated the presence of a splicing variant caused by the combined loss of exons 5 and 6 and direct splicing of exon 4 with 7. d) RON splice variant lacking exon 6 in cell line H358. This indicated the presence of a splicing variant caused by loss of exon 6 and direct splicing of exon 5 with 7. e) RON splice variant caused by combined loss of exons 8 and 9 in cell line SW900. This indicated the presence of only the splicing variant resulting from the combined loss of exons 8 and 9 and direct splicing of exon 7 with 10

Fig. 2

Schematic diagram of splice variants of extracellular coding region of RON. a) Two juxtaposed Y1238 and Y1239 in the kinase domain; phosphorylation sites Y1353 and Y1360 in the C-terminal docking site for multiple substrates with src homology 2 (SH2) domain; the other important motifs/domains are secretory peptide signal sequence, sema, plexin, semaphorin and integrin (PSI), Ig-like, plexins, transcription factors (IPT), transmembrane (TM) and tyrosine kinase (TK). b) 20 coding exons of RON shown in proportion to sequence length. c) 1187 bps PCR amplicon sequenced in the present study. d) 789 bps PCR amplicon sequenced in the present study. e) Specific, skipped exons by RON cDNA in lung cancer cell lines, as identified by PCR amplification and sequencing in the present study are: exon 2 (189 bps); exons 2 + 3 (318 bps); exons 5 + 6 (327 bps); exon 6 (166 bps) and exons 8 + 9 (256 bps)