Krishnaswamy Soundararajan, Mohammed Abdul Khader, Amer Osama E, Tripathi Gyanendra, Alokail Majed S, Al-Daghri Nasser M
Biomarkers Research Program, Riyadh Biochemistry Department, College of Science, King Saud University, 11451 Riyadh, Saudi Arabia; Prince Mutaib Chair for Biomarkers of Osteoporosis, Riyadh Biochemistry Department, College of Science, King Saud University, 11451 Riyadh, Saudi Arabia.
Division of Metabolic and Vascular Research, University of Warwick, Coventry CV2 2DX, UK.
Lung Cancer. 2016 Feb;92:41-6. doi: 10.1016/j.lungcan.2015.12.002. Epub 2015 Dec 12.
Altered expressions of receptor tyrosine kinases drive the growth and metastasis of several cancers. RON is a single pass transmembrane receptor tyrosine kinase (RTK) shown to be aberrantly expressed in various cancer types. However, target validation and successful therapeutic targeting of RON in cancers is hampered by the co-existence of unknown number/types of isoforms, which are structurally similar but functionally diverse.
The objective of this study was to identify differential splicing in the C-terminal region of RON transcripts to better understand RON signaling in cancers. mRNA transcript sequence between exons 14 and 20 of RON was PCR amplified and sequenced using cDNA from 10 SCLC and 13 NSCLC cell lines. Specific exon deletions were identified by aligning sequencing chromatograms with reference RON cDNA sequence.
We identified the presence of four unique transcript sequence variants of RON formed through skipping of exons 15-19, 16-19, 16-17 and 16. The transcript variants, except the one lacking exons 15-19, were found in more than one cell line. Several cell lines contained two to four of these uniquely spliced transcript variants. dbEST (Expressed Sequence Tags database) or other DNA sequence databases did not contain RON cDNA sequences corresponding to any of the above exon deletions indicating that all these transcript sequence alterations are novel.
Results of our study indicate common occurrence of different types of alternatively spliced transcripts of RON in lung cancer with potential to be translated into proteins lacking active kinase domain. Our findings suggest that tumors produce several dominant negative isoforms which probably inhibit ligand dependent RON signaling, and hence, raise important questions regarding the appropriateness of blocking wild type RON signaling for therapy. Further, presence of transcript variants and their isoform products may interfere with quantitative and functional analysis during target validation.
受体酪氨酸激酶的表达改变驱动多种癌症的生长和转移。RON是一种单次跨膜受体酪氨酸激酶（RTK），已证实在多种癌症类型中异常表达。然而，由于存在数量和类型未知的异构体，它们在结构上相似但功能多样，这阻碍了对RON在癌症中的靶点验证和成功的治疗靶向。
本研究的目的是鉴定RON转录本C末端区域的差异剪接，以更好地理解癌症中的RON信号传导。使用来自10个小细胞肺癌（SCLC）和13个非小细胞肺癌（NSCLC）细胞系的cDNA，对RON外显子14和20之间的mRNA转录本序列进行PCR扩增和测序。通过将测序色谱图与参考RON cDNA序列比对来鉴定特定外显子缺失。
我们鉴定出通过跳过外显子15 - 19、16 - 19、16 - 17和16形成的四种独特的RON转录本序列变体。除了缺少外显子15 - 19的转录本变体，其他变体在不止一个细胞系中被发现。几个细胞系包含两到四种这些独特剪接的转录本变体。dbEST（表达序列标签数据库）或其他DNA序列数据库中不包含与上述任何外显子缺失相对应的RON cDNA序列，表明所有这些转录本序列改变都是新的。
我们的研究结果表明，不同类型的RON可变剪接转录本在肺癌中普遍存在，有可能被翻译成缺乏活性激酶结构域的蛋白质。我们的发现表明肿瘤产生几种显性负性异构体，可能抑制配体依赖性RON信号传导，因此，引发了关于阻断野生型RON信号传导用于治疗的适宜性的重要问题。此外,转录本变体及其异构体产物的存在可能会干扰靶点验证过程中的定量和功能分析。
