Exploiting endocytic factors as druggable targets to enhance sodium iodide symporter activity with clinical implications for radioiodide therapy

Katie Brookes; Ling Zha; Jana Kim; Merve Kocbiyik; Selvambigai Manivannan; Kavitha Sunassee; Philip Blower; Hannah Nieto; Vicki Smith; Martin Read; Christopher McCabe

doi:10.1186/s13044-023-00170-8

Abstract

Background: Suboptimal radioiodide (RAI) treatment is frequently associated with diminished targeting and retention of the sodium iodide symporter (NIS) at the plasma membrane (PM). The mechanisms which govern the endocytosis of NIS away from the PM are however ill-defined and may have therapeutic potential. We recently demonstrated that NIS internalisation was modulated by the interaction of a C-terminal diacidic motif with the heterotetramer Adaptor Protein 2 (AP2) – a key regulatory factor in endocytosis. Here, we determined whether NIS endocytosis represents a druggable process to enhance RAI uptake.

Methods: PM localisation of NIS was quantified via NanoBRET and cell surface biotinylation assays (CSBA). RAI (125I) uptake assays were used to monitor NIS function in vitro. Intravenous technetium-99m pertechnetate (99mTc) uptake was used to evaluate NIS function in wild-type BALB/c mice.

Results: The drug chloroquine (CQ) rapidly increased 125I uptake in TPC1-NIS (2.54-fold; Ppeaking after 8 hr, which was abrogated by co-treatment with the endocytosis inhibitor Dynasore. Subsequent CSBA confirmed elevated levels of cell-surface NIS in CQ-treated thyroid cancer cells. This finding was supported in live CQ-treated cells via KRAS-NanoBRET assays, where CQ gave a strong BRET signal similar to Dynasore, suggesting that NIS was retained at the PM. To challenge this, we ablated PICALM, an endocytic factor known to recruit AP2/clathrin to the PM which prevented significant induction of RAI uptake by CQ. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal 99mTc-uptake in combination with the HDACi SAHA (52.7%; n = 10; P = 0.0003), as well as increasing thyroidal expression of NIS (2.2-fold; P < 0.0001), TSHR (1.9-fold; P = 0.001) and PAX8 mRNA (1.6-fold; P = 0.003).

Conclusions: Our findings suggest that CQ interferes with the PICALM/AP2/clathrin machinery which controls NIS endocytosis, identifying it as an FDA-approved pharmaceutical agent which alters NIS endocytosis, with translatable

Original language	English
Article number	OR4
Pages (from-to)	4-4
Number of pages	1
Journal	Thyroid Research
Volume	16
Issue number	Supplement 1
DOIs	https://doi.org/10.1186/s13044-023-00170-8
Publication status	Published - 11 Jul 2023
Event	71st Annual Meeting of the British Thyroid Association - Royal College of Pathologists, London, United Kingdom Duration: 9 Jun 2023 → 9 Jun 2023 Conference number: 71

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Making Radioiodine Treatment a Realistic Therapeutic Opportunity in Breast Cancer
McCabe, C.
US ARMY
30/09/21 → 29/09/25
Project: Research

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Brookes, K., Zha, L., Kim, J., Kocbiyik, M., Manivannan, S., Sunassee, K., Blower, P., Nieto, H., Smith, V., Read, M., & McCabe, C. (2023). Exploiting endocytic factors as druggable targets to enhance sodium iodide symporter activity with clinical implications for radioiodide therapy. Thyroid Research, 16(Supplement 1), 4-4. Article OR4. https://doi.org/10.1186/s13044-023-00170-8

@article{b6453e76ab954318bb785e292d7019b7,

title = "Exploiting endocytic factors as druggable targets to enhance sodium iodide symporter activity with clinical implications for radioiodide therapy",

abstract = "Background: Suboptimal radioiodide (RAI) treatment is frequently associated with diminished targeting and retention of the sodium iodide symporter (NIS) at the plasma membrane (PM). The mechanisms which govern the endocytosis of NIS away from the PM are however ill-defined and may have therapeutic potential. We recently demonstrated that NIS internalisation was modulated by the interaction of a C-terminal diacidic motif with the heterotetramer Adaptor Protein 2 (AP2) – a key regulatory factor in endocytosis. Here, we determined whether NIS endocytosis represents a druggable process to enhance RAI uptake. Methods: PM localisation of NIS was quantified via NanoBRET and cell surface biotinylation assays (CSBA). RAI (125I) uptake assays were used to monitor NIS function in vitro. Intravenous technetium-99m pertechnetate (99mTc) uptake was used to evaluate NIS function in wild-type BALB/c mice. Results: The drug chloroquine (CQ) rapidly increased 125I uptake in TPC1-NIS (2.54-fold; Ppeaking after 8 hr, which was abrogated by co-treatment with the endocytosis inhibitor Dynasore. Subsequent CSBA confirmed elevated levels of cell-surface NIS in CQ-treated thyroid cancer cells. This finding was supported in live CQ-treated cells via KRAS-NanoBRET assays, where CQ gave a strong BRET signal similar to Dynasore, suggesting that NIS was retained at the PM. To challenge this, we ablated PICALM, an endocytic factor known to recruit AP2/clathrin to the PM which prevented significant induction of RAI uptake by CQ. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal 99mTc-uptake in combination with the HDACi SAHA (52.7%; n = 10; P = 0.0003), as well as increasing thyroidal expression of NIS (2.2-fold; P < 0.0001), TSHR (1.9-fold; P = 0.001) and PAX8 mRNA (1.6-fold; P = 0.003). Conclusions: Our findings suggest that CQ interferes with the PICALM/AP2/clathrin machinery which controls NIS endocytosis, identifying it as an FDA-approved pharmaceutical agent which alters NIS endocytosis, with translatable ",

author = "Katie Brookes and Ling Zha and Jana Kim and Merve Kocbiyik and Selvambigai Manivannan and Kavitha Sunassee and Philip Blower and Hannah Nieto and Vicki Smith and Martin Read and Christopher McCabe",

year = "2023",

month = jul,

day = "11",

doi = "10.1186/s13044-023-00170-8",

language = "English",

volume = "16",

pages = "4--4",

journal = "Thyroid Research",

issn = "1756-6614",

publisher = "BioMed Central",

number = "Supplement 1",

note = "71st Annual Meeting of the British Thyroid Association<br/>, Annual BTA Meeting ; Conference date: 09-06-2023 Through 09-06-2023",

}

Brookes, K, Zha, L, Kim, J, Kocbiyik, M, Manivannan, S, Sunassee, K, Blower, P, Nieto, H , Smith, V , Read, M & McCabe, C 2023, 'Exploiting endocytic factors as druggable targets to enhance sodium iodide symporter activity with clinical implications for radioiodide therapy', Thyroid Research, vol. 16, no. Supplement 1, OR4, pp. 4-4. https://doi.org/10.1186/s13044-023-00170-8

TY - JOUR

T1 - Exploiting endocytic factors as druggable targets to enhance sodium iodide symporter activity with clinical implications for radioiodide therapy

AU - Brookes, Katie

AU - Zha, Ling

AU - Kim, Jana

AU - Kocbiyik, Merve

AU - Manivannan, Selvambigai

AU - Sunassee, Kavitha

AU - Blower, Philip

AU - Nieto, Hannah

AU - Smith, Vicki

AU - Read, Martin

AU - McCabe, Christopher

N1 - Conference code: 71

PY - 2023/7/11

Y1 - 2023/7/11

N2 - Background: Suboptimal radioiodide (RAI) treatment is frequently associated with diminished targeting and retention of the sodium iodide symporter (NIS) at the plasma membrane (PM). The mechanisms which govern the endocytosis of NIS away from the PM are however ill-defined and may have therapeutic potential. We recently demonstrated that NIS internalisation was modulated by the interaction of a C-terminal diacidic motif with the heterotetramer Adaptor Protein 2 (AP2) – a key regulatory factor in endocytosis. Here, we determined whether NIS endocytosis represents a druggable process to enhance RAI uptake. Methods: PM localisation of NIS was quantified via NanoBRET and cell surface biotinylation assays (CSBA). RAI (125I) uptake assays were used to monitor NIS function in vitro. Intravenous technetium-99m pertechnetate (99mTc) uptake was used to evaluate NIS function in wild-type BALB/c mice. Results: The drug chloroquine (CQ) rapidly increased 125I uptake in TPC1-NIS (2.54-fold; Ppeaking after 8 hr, which was abrogated by co-treatment with the endocytosis inhibitor Dynasore. Subsequent CSBA confirmed elevated levels of cell-surface NIS in CQ-treated thyroid cancer cells. This finding was supported in live CQ-treated cells via KRAS-NanoBRET assays, where CQ gave a strong BRET signal similar to Dynasore, suggesting that NIS was retained at the PM. To challenge this, we ablated PICALM, an endocytic factor known to recruit AP2/clathrin to the PM which prevented significant induction of RAI uptake by CQ. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal 99mTc-uptake in combination with the HDACi SAHA (52.7%; n = 10; P = 0.0003), as well as increasing thyroidal expression of NIS (2.2-fold; P < 0.0001), TSHR (1.9-fold; P = 0.001) and PAX8 mRNA (1.6-fold; P = 0.003). Conclusions: Our findings suggest that CQ interferes with the PICALM/AP2/clathrin machinery which controls NIS endocytosis, identifying it as an FDA-approved pharmaceutical agent which alters NIS endocytosis, with translatable

AB - Background: Suboptimal radioiodide (RAI) treatment is frequently associated with diminished targeting and retention of the sodium iodide symporter (NIS) at the plasma membrane (PM). The mechanisms which govern the endocytosis of NIS away from the PM are however ill-defined and may have therapeutic potential. We recently demonstrated that NIS internalisation was modulated by the interaction of a C-terminal diacidic motif with the heterotetramer Adaptor Protein 2 (AP2) – a key regulatory factor in endocytosis. Here, we determined whether NIS endocytosis represents a druggable process to enhance RAI uptake. Methods: PM localisation of NIS was quantified via NanoBRET and cell surface biotinylation assays (CSBA). RAI (125I) uptake assays were used to monitor NIS function in vitro. Intravenous technetium-99m pertechnetate (99mTc) uptake was used to evaluate NIS function in wild-type BALB/c mice. Results: The drug chloroquine (CQ) rapidly increased 125I uptake in TPC1-NIS (2.54-fold; Ppeaking after 8 hr, which was abrogated by co-treatment with the endocytosis inhibitor Dynasore. Subsequent CSBA confirmed elevated levels of cell-surface NIS in CQ-treated thyroid cancer cells. This finding was supported in live CQ-treated cells via KRAS-NanoBRET assays, where CQ gave a strong BRET signal similar to Dynasore, suggesting that NIS was retained at the PM. To challenge this, we ablated PICALM, an endocytic factor known to recruit AP2/clathrin to the PM which prevented significant induction of RAI uptake by CQ. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal 99mTc-uptake in combination with the HDACi SAHA (52.7%; n = 10; P = 0.0003), as well as increasing thyroidal expression of NIS (2.2-fold; P < 0.0001), TSHR (1.9-fold; P = 0.001) and PAX8 mRNA (1.6-fold; P = 0.003). Conclusions: Our findings suggest that CQ interferes with the PICALM/AP2/clathrin machinery which controls NIS endocytosis, identifying it as an FDA-approved pharmaceutical agent which alters NIS endocytosis, with translatable

U2 - 10.1186/s13044-023-00170-8

DO - 10.1186/s13044-023-00170-8

M3 - Abstract

SN - 1756-6614

VL - 16

SP - 4

EP - 4

JO - Thyroid Research

JF - Thyroid Research

IS - Supplement 1

M1 - OR4

T2 - 71st Annual Meeting of the British Thyroid Association<br/>

Y2 - 9 June 2023 through 9 June 2023

ER -

Exploiting endocytic factors as druggable targets to enhance sodium iodide symporter activity with clinical implications for radioiodide therapy

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Making Radioiodine Treatment a Realistic Therapeutic Opportunity in Breast Cancer

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