Differential roles of RET isoforms in medullary and papillary thyroid carcinomas

Abstract

The RET receptor tyrosine kinase mediates cell proliferation, survival and migration in embryogenesis and is implicated in the transformation and tumour progression in multiple cancers. RET is frequently mutated and constitutively activated in familial and sporadic thyroid carcinomas. As a result of alternative splicing, RET is expressed as two protein isoforms, RET9 and RET51, which differ in their unique C-terminal amino acids. These isoforms have distinct intracellular trafficking and associated signalling complexes, but functional differences are not well defined. We used shRNA-mediated knockdown (KD) of individual RET isoforms or of total RET to evaluate their functional contributions in thyroid carcinoma cells. We showed that RET is required for cell survival in medullary (MTC) but not papillary thyroid carcinoma (PTC) cells. In PTC cells, RET depletion reduced cell migration and induced a flattened epithelial-like morphology. RET KD decreased the expression of mesenchymal markers and matrix metalloproteinases and reduced anoikis resistance and invasive potential. Further, we showed that RET51 depletion had significantly greater effects on each of these processes than RET9 depletion in both MTC and PTC cells. Finally, we showed that expression of RET, particularly RET51, was correlated with malignancy in a panel of human thyroid tumour tissues. Together, our data show that RET expression promotes a more mesenchymal phenotype with reduced cell-cell adhesion and increased invasiveness in PTC cell models, but is more important for tumour cell survival, proliferation and anoikis resistance in MTC models. Our data suggest that the RET51 isoform plays a more prominent role in mediating these processes compared to RET9.

Exploiting Ret Isoforms in Managing Medullary and Papillary Thyroid Cancer

“…In recent studies, we have shown that RET51 modulates multiple critical processes that promote tumor growth and invasive spread in thyroid carcinomas [21]. In both MTC and PTC cell types, RET51 is significantly more effective than RET9 in enhancing cell proliferation, migration and anchorage-independent growth, suggesting that it may be the primary isoform contributing to tumor aggressiveness and disease progression [21]. Consistent with this, RET51 expression is correlated with increased malignancy in human thyroid tumors [21].…”

“…Despite the abundance of evidence for distinct roles of the RET isoforms, the implications of RET isoform expression in cancer, and its potential as a marker of disease course, have not been fully explored. In recent studies, we have shown that RET51 modulates multiple critical processes that promote tumor growth and invasive spread in thyroid carcinomas [21]. In both MTC and PTC cell types, RET51 is significantly more effective than RET9 in enhancing cell proliferation, migration and anchorage-independent growth, suggesting that it may be the primary isoform contributing to tumor aggressiveness and disease progression [21].…”

“…In both MTC and PTC cell types, RET51 is significantly more effective than RET9 in enhancing cell proliferation, migration and anchorage-independent growth, suggesting that it may be the primary isoform contributing to tumor aggressiveness and disease progression [21]. Consistent with this, RET51 expression is correlated with increased malignancy in human thyroid tumors [21]. RET51 is also more highly expressed relative to RET9 in some MEN2-associated tumors [22] and in breast carcinomas, where it may grant a growth advantage [23].…”

RET-mediated modulation of tumor microenvironment and immune response in multiple endocrine neoplasia type 2 (MEN2)

“…Accordingly, treatment of an MTC cell line expressing the RET MEN2A mutant allele (TT) with sunitinib, a RET kinase inhibitor, inhibited EMT-related gene expression (Jain et al 2004b, Ameur et al 2009). RET51-and RET9-specific RET depletion in TT cells, obtained by using RNAi or a RET kinase-dead (RetKD) mutant, caused a drop in the expression of EMT transcription factors ZEB1 and TWIST1 (Lian et al 2017) and inhibited EMT-related activities (anoikis resistance, anchorage-independent growth and invasion). Moreover, this approach demonstrated that RET51 was more efficient than RET9 in sustaining EMT-related activities (Lian et al 2017).…”

Differential recruitment of E3 ubiquitin ligase complexes regulates RET isoform internalization

“…1A). RET isoforms are generally coexpressed, but differ in their abilities to promote cell growth, motility and invasion (Lian et al, 2017). RET interactions with protein adaptors and signalling molecules are predominantly mediated through the key isoformspecific phosphorylated tyrosine residues pY1062 (in RET9) and pY1096 (in RET51) and a C-terminal PDZ-binding motif (FTRF 1072 in RET9 only) that lie within these isoform-specific tails (Mulligan, 2014).…”