Characterization of isoform specific RET knockdown in cancer cell lines
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The REarranged in Transfection (RET) tyrosine kinase is an important signalling protein for the development of neural crest-derived tissues such as the enteric and sympathetic nervous systems. RET is constitutively activated in multiple human tumour types, such as thyroid carcinomas and some non-small cell lung cancers. RET has 3 distinct isoforms, RET9, RET43 and RET51, which are named after the lengths of their unique C-terminal tails. Here, we investigate the role of RET in the TT thyroid carcinoma cell line, where it is a driver of tumourigenesis, and in the MiaPaCa-2 pancreatic carcinoma cell line, where RET is not driving tumour initiation, but may nonetheless have a profound effect on tumour progression. We generated lentiviral constructs for shRNAs that target either RET9 or RET51 specifically, or a common region shared by all RET isoforms. TT and MiaPaCa-2 cells were transduced using these lentiviral particles to create stable cell lines containing knockdowns of total RET, RET9, or RET51. Using a variety of morphological and biochemical assays, we found that RET expression is critical for TT cell survival, and that both RET9 and RET51 play significant roles in driving cell proliferation in TT cells. Conversely, RET is not critical for MiaPaCa-2 cell survival, and RET knockdown had no effect on MiaPaCa-2 proliferation. MiaPaCa-2 cells instead underwent dramatic morphological changes, from their normal spindle-like mesenchymal appearance to an increasingly flattened and epithelioid character, in response to RET9, RET51 or total-RET knockdown. The observed morphological changes were coupled with significantly reduced invasiveness through matrigel towards a source of chemoattractant, suggesting a critical role for RET in mediating cell invasiveness. These results suggest that RET may not only drive tumourigenesis, but can also enhance disease progression when expressed in other tumour types. We predict that RET may play critical roles in perineural invasion in pancreatic cancers, a process where cells invade along peripheral nerve fibers by following an increasing concentration of chemoattractants secreted by nerve and glial cells. Thus, RET may be a valuable target to slow, or stop this process, which would have significant clinical implications in a wide variety of cancers.