Higher RET Gene Expression Levels Do Not Represent anAlternative RET Activation Mechanism in Medullary Thyroid Carcinoma

Abstract

This study was designed to investigate whether RET (rearranged during transfection) mRNA over-expression could be considered an alternative driver event for the development of medullary thyroid carcinoma (MTC), and if different RET isoforms could play a role in MTC tumorigenesis. Eighty-three MTC patients, whose mutational profile was previously identified by next-generation sequencing (NGS) IONS5, were included in this study. Expression analysis was performed by the quantitative reverse transcription-polymerase chain reaction technique. RET expression levels were found to be significantly higher in cases with RET somatic mutations than in cases that were negative for RET somatic mutations (p = 0.003) as well as in cases with a somatic mutation, either in RET or RAS than in cases negative for both these mutations (p = 0.01). All cases were positive for the RET51 isoform expression while only 72/83 (86.7%) were positive for RET9 isoform expression. A statistically significant higher expression of the RET51 isoform was found in cases positive for RET somatic mutation than in cases either positive for RAS mutation (p = 0.0006) or negative for both mutations (p = 0.001). According to our data, RET gene over-expression does not play a role in MTC tumorigenesis, neither as an entire gene or as an isoform. At variance, the RET gene, and in particular the RET51 isoform, is expressed higher in RET mutated cases. On the basis of these results we can hypothesize that the overexpression of RET, and in particular of RET51, could potentiate the transforming activity of mutated RET, making these cases more aggressive.

RET splice site variants in medullary thyroid carcinoma

“…The RET proto-oncogene encodes a receptor tyrosine kinase that when activated either by a point mutation or gene rearrangement can result in a constitutively active cytosolic oncoprotein ( Mulligan, 2014 ; Chakravarty et al, 2017 ). Although RET mutations are well described in MTC ( Ciampi et al, 2019 ), there is a paucity of information about RET splice variants in MTC, though one study of a MTC cell line described three splice variants, RET 2/4, RET 2/5, and RET 2/6 and another described functional isoforms RET51 and RET9 ; however, neither played a role in MTC tumorigenesis ( Lorenzo et al, 1995 ; Mule et al, 2021 ). Our results demonstrate that irrespective of a driving mutation, there is a high frequency of RET splice variants in MTC, with all 25 MTCs in this study harboring at least one RET splice variant versus only 0.3% of 3,599 non-MTCs.…”

“…The authors concluded that wild-type and alternatively spliced RET transcripts co-exist with rearrangements in PTC and may play a role in thyroid tumorigenesis. In MTC though, high RET gene expression levels have not been associated with an alternative RET activation mechanism ( Mule et al, 2021 ).…”

“…These RET isoforms displayed unique levels of auto-phosphorylation and had differential interactions with adaptor proteins and alternative splicing in intron 19 ( Richardson et al, 2012 ; Ramone et al, 2019 ). These isoforms also displayed distinct subcellular localizations, trafficking properties, and downstream signaling; however, neither was demonstrated to play a role in MTC tumorigenesis ( Mule et al, 2021 ).…”

“…It has been demonstrated that while MTCs overexpress RET mRNA this overexpression does not play a significant role in tumorigenesis ( Lian et al, 2017 ; Mule et al, 2021 ). It is possible that RET 2/4 and 2/8 are detected secondary to RET mRNA overexpression and play no significant role in tumorigenesis.…”

“…Two functional isoforms, RET51 and RET9 , formed via alternative splicing near the C-terminus (3’ end) of RET have been found to play distinct roles in tumorigenesis and/or development ( Richardson et al, 2012 ). Furthermore, both RET51 and RET9 functional isoforms have been identified in MTC; however, neither played a role in MTC tumorigenesis ( Mule et al, 2021 ). Lorenzo et al described three alternative splice variants in a MTC cell line ( Lorenzo et al, 1995 ).…”