By exploiting the canonical function of the sodium iodide symporter
(NIS), ablative radioiodide therapy is an effective treatment for thyroid
cancer and has been hypothesised as a viable treatment for
breast cancer. However, up to a quarter of patients are unable to
accumulate sufficient radioiodide for effective treatment due to
decreased expression of NIS and/or reduced plasma membrane
localisation. Currently, the regulation of NIS trafficking and its localisation
at the plasma membrane is ill-defined.
To identify novel NIS-interactors, and unravel the mechanism of NIS
trafficking, mass spectrometry analysis was performed on proteins
co-immunoprecipitating with lentivirally expressed NIS in cell plasma
membrane extracts. To determine the biological impact of these
putative interactors on NIS, siRNA knockdown of the top shortlisted
interactors was followed by radioiodide uptake assays. Further, to
validate interactors that altered NIS function, co-immunoprecipitation
and proximity ligation assays were completed.
NIS activity was significantly altered by ADP-ribosylation factor 4
(ARF4) and valosin containing protein (VCP) in thyroid and breast
cancer cell lines stably-expressing NIS. ARF4 downregulation significantly
decreased radioiodide uptake by 75% and 44%, and VCP
downregulation increased radioiodide uptake by 71% and 56%, in
the thyroid and breast cell lines, respectively. In contrast, ARF4 overexpression
significantly increased radioiodide uptake by 89% and
43%, and VCP overexpression decreased radioiodide uptake by 52%
and 38%, in thyroid and breast cell lines, respectively. Through both
co-immunoprecipitation and proximity ligation assays it was confirmed
that NIS interacts with ARF4 and VCP. Analysis of TCGA data
from N = 58 matched papillary thyroid cancers revealed ARF4 is significantly
repressed and VCP highly upregulated in thyroid cancer,
providing a new putative explanation for repressed NIS function.
These studies elucidating the regulation of NIS localisation have identified
two novel potential therapeutic targets for enhancing radioiodide
uptake in patients who are radioiodide-refractory.