The most common form of endocrine cancer is differentiated thyroid cancer (DTC). Outcomes of DTC largely depend on radioiodine treatment, which is mediated the sodium-iodide symporter (NIS). However, many tumours exhibit NIS dysregulation, resulting in a poorer prognosis. Since breast cancer can also overexpress NIS, albeit of limited function, radioiodine treatment may be a promising treatment option. Our previous data show that overexpression of the pituitary tumor-transforming gene-binding factor (PBF) is partially responsible for the reduced function of NIS in thyroid and breast cancer. The interaction of PBF with NIS leads to an alteration of NIS localisation away from the plasma membrane. Binding of NIS requires a C-terminal PBF tyrosine residue 174 (Y174) to be phosphorylated by the tyrosine kinase Src. To address the mechanistic interactions between NIS, PBF and Src we used CRISPR/Cas9 to knock PBF out in TPC1 and Nthy-ori thyroid cells, as well as in MDA-MB-231 and MCF7 breast cell lines. Endonuclease screening, Western blotting and DNA sequencing identified successful PBF knock out with at least two different guide RNAs (gRNA) in TPC1 and N-thy-ori thyroid CRISPR cell lines. Knockout of PBF in TPC1s was associated with a 50.33% (gRNA#1) and 49.13% (gRNA#2) increase in radioiodide uptake compared to parental lines expressing NIS, whilst Nthy-ori cells showed a 75.04% (gRNA#1) and 45.12% (gRNA#3) increase. Transfection of Src into CRISPR- PBF versus parental lines resulted in a similar magnitude of radioiodide uptake repression. Thus, PBF is directly implicated in the intrinsic activity of NIS in vitro, but it is likely that Src phosphorylates additional targets to PBF, which are also able to directly or indirectly repress iodide uptake.
|Number of pages||1|
|Publication status||Published - 1 Nov 2018|
|Event||Society for Endocrinology BES 2018 - Scottish Event Campus (SEC), Glasgow, United Kingdom|
Duration: 19 Nov 2018 → 21 Nov 2018