Abstract
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Objectives: Canonical role of thyrotropin (TSH) -TSH receptor (TSHR) signaling is regulating iodide uptake and thyroid hormone bio-synthesis, making it necessary to elevate serum TSH levels before radioiodide (131I) therapy for differentiated thyroid cancer (DTC) by T4 withdraw or rTSH administration. However, non-iodide avid metastases of DTC appear not to response to stimulation of high levels of TSH, suggesting potential mis-transduction of downstream of TSH-TSHR signaling. PI3K/AKT/mTOR signaling activation has been shown to be associated with decreased iodide uptake with the mechanism remaining elusive. Therefore, in this study, we aim to explore the role of non-canonical TSH-TSHR signaling in regulating cell motility and iodide uptake function in thyroid cancers in relationship with PI3K/AKT/mTOR signaling. Methods: We utilized human thyroid cancer cell lines TPC1, BCPAP and FTC-133 and a rat thyroid cell line PCCL3. Co-Immunoprecipitation was performed from total lysate of thyroid cancer cells to observe the interaction between Gα12/13 and leukemia associated RhoA guanine exchange factor (LARG). RhoA pull-down activation assay was utilized to detect activated RhoA-GTP levels and wound healing assay was performed to observe cell migration rate. Through siRNA knockdown, we investigated the role of Gα12/13 and LARG in TSHR signaling. Active mutation Gα12Q229L or Gα13Q226L was transiently and stably transfected into TPC1 cell to further explore the role of Gα12/13 in regulating cell motility and differentiation. PTEN siRNA knockdown, PTEN transiently transfection and PI3K/AKT/mTOR inhibitors were utilized to study the crosstalk between PI3K/AKT/mTOR signaling and downstream of TSHR signaling.
Results: We found that in thyroid cancer cells, TSH binding to TSHR coupled to non-canonical Gα12/13 protein, then to activate RhoA through interaction with LARG, resulting in pro-migration tumorigenic phenotype. Gα12/13 activation also results in decreased thyroid specific molecular expression including TTF1, PAX8, TPO, Tg and NIS as well as iodide uptake, probably by inhibiting downstream of Gαs signaling. PI3K/AKT/mTOR signaling activation ehances Gα12/13 signaling by increasing LARG levels while it is also involved in inhibiting downstream of Gαs signaling.
Conclusions: Our results elucidate non-canonical activation of TSH-TSHR signaling and its role in increasing cell mobility and dedifferentiation in thyroid cancer through crosstalk with PI3K/AKT/mTOR signaling.
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