HMGB1-mediated autophagy regulates sodium/iodide symporter protein degradation in thyroid cancer cells

Abstract

Background: Sodium/iodide symporter (NIS)-mediated iodide uptake plays an important physiological role in regulating thyroid gland function, as well as in diagnosing and treating Graves' disease and thyroid cancer. High-mobility group box 1 (HMGB1), a highly conserved nuclear protein, is a positive regulator of autophagy conferring resistance to chemotherapy, radiotherapy and immunotherapy in cancer cells. Here the authors intended to identify the role of HMGB1 in Hank's balanced salt solution (HBSS)-induced autophagy, explore NIS protein degradation through a autophagy-lysosome pathway in thyroid cancer cells and elucidate the possible molecular mechanisms.

Methods: Immunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR) were performed for detecting the expression of HMGB1 in different tissues. HMGB1 was knocked down by lentiviral transfection in FTC-133/TPC-1 cells. Autophagic markers LC3-II, p62, Beclin1 and autophagosomal formation were employed for evaluating HMGB1-mediated autophagy in HBSS-treated cells by Western blot, immunofluorescence and electron microscopy. Western blot, quantitative RT-PCR and gamma counter analysis were performed for detecting NIS expression and iodide uptake in HMGB1-knockdown cells after different treatments. The reactive oxygen species (ROS) level, ROS-mediated LC3-II expression and HMGB1 cytosolic translocation were detected by fluorospectrophotometer, flow cytometry, Western blot and immunofluorescence. HMGB1-mediated AMPK, mTOR and p70S6K phosphorylation (p-AMPK, p-mTOR & p-p70S6K) were detected by Western blot. Furthermore, a nude murine model with transplanted tumor was employed for examining the effect of HMGB1-mediated autophagy on imaging and biodistribution of ^99mTcO4^-. NIS, Beclin1, p-AMPK and p-mTOR were detected by immunohistochemical staining and Western blot in transplanted tumor samples.

Results: HMGB1 was a critical regulator of autophagy-mediated NIS degradation in HBSS-treated FTC-133/TPC-1 cells. And HMGB1 up-regulation was rather prevalent in thyroid cancer tissues and closely correlated with worse overall lymph node metastasis and clinical stage. HMGB1-knockdown dramatically suppressed autophagy, NIS degradation and boosted iodide uptake in HBSS-treated cells. Moreover, HBSS enhanced ROS-sustained autophagy and promoted the cytosolic translocation of HMGB1. A knockdown of HMGB1 suppressed LC3-II conversion and NIS degradation via an AMPK/mTOR-dependent signal pathway through a regulation of ROS generation, rather than ATP. Furthermore, these data were further supported by our in vivo experiment of xenografts formed by HMGB1 knockdown cells reverting the uptake of ^99mTcO4^- as compared with control shRNA-transfected cells in hunger group.

Conclusions: Acting as a critical regulator of autophagy-mediated NIS degradation via ROS/AMPK/mTOR pathway, HMGB1is a potential intervention target of radioiodine therapy in thyroid cancer.

Keywords: AMPK; Autophagy; HMGB1; NIS; mTOR.

Fig. 1

HMGB1 expression became up-regulated in thyroid cancer and was associated with clinicopathologic features (a) Western blot of HMGB1 and actin in various cell lines hinted at an over-expression of HMGB1 in thyroid cancer; (b) Immunohistochemical staining of HMGB1 was performed for different tissues. TC, thyroid cancer; TA, thyroid adenoma; SG, simple goiter; N, normal thyroid; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; (c) Relative expression levels of HMGB1 in different tissues. Total mRNA was extracted from normal or patient tissues and HMGB1 level determined by relative optical intensity (in arbitrary units, AU) of bands on RT-PCR. Each dot represented relative level of HMGB1 in each individual sample. *P < 0.01 vs. normal thyroid; **P > 0.01 vs. normal thyroid; #P > 0.01 vs. normal thyroid; (d) Relative expression levels of HMGB1 in thyroid cancer. Total mRNA was extracted from thyroid cancer patients’ tissues and HMGB1 level determined by relative optical intensity (in arbitrary units, AU) of bands on RT-PCR. Each dot represented relative level of HMGB1 in an individual sample. *P > 0.01 vs. FTC; (e) HMGB1 expression level for differentiating thyroid cancer tissues from non-thyroid cancer tissues in our validated cohort; AUC: 96.7%, sensitivity: 88.9% and specificity: 96.2% in the validated cohort

Fig. 2

HMGB1 regulated autophagy in thyroid cancer cells (a) FTC-133/TPC-1 cells were transfected with HMGB1 shRNA and control shRNA and then starved by HBSS for 2 h. And LC3-I/II level was assayed by Western blot; (b) FTC-133/TPC-1 cells were transfected with HMGB1 shRNA and control shRNA and then pre-treated for 1 h with pepstatin A (PA, 10 μM) and E64D (10 μM) as indicated. Cells were subsequently treated for 3 h with HBSS in continuous presence or absence PA/E64D inhibitors. LC3-I/II, Beclin1 and p62 levels were assayed by Western blot; (c) Ultrastructural features in FTC-133/TPC-1 cells transfected with HMGB1 shRNA and control shRNA after a 3-h treatment of HBSS. More autophagosomes were seen in control shRNA plus HBSS-treated cells than in cells treated with HMGB1 shRNA plus HBSS. Arrows indicated autophagosomes

Fig. 3

HMGB1-mediated autophagy regulated NIS expression and iodide uptake (a). FTC-133/TPC-1 cells were pretreated with 3-MA (10 mM) treatment for 1 h and then starved by HBSS for 3 h. LC3-I/II and NIS levels were assayed by Western blot; (b&c) FTC-133/TPC-1 cells were transfected with HMGB1 shRNA and control shRNA and then starved by HBSS for 3 h. LC3-I/II and NIS levels were assayed by Western blot. And NIS mRNA was assayed by qRT-PCR (n = 3, *P > 0.01, **P > 0.01); (d) Dynamic uptaking: FTC-133/TPC-1 cells were transfected with HMGB1 shRNA and control shRNA and starved by HBSS for 3 h. Indicated cells were cultured with 175 KBq ¹³¹I for 5, l0, 20, 30, 60, 90 and 120 min. The uptake of ¹³¹I in indicated cells was detected by a gamma counter; (e) Radionuclide uptaking: FTC-133/TPC-1 cells were transfected with HMGB1 shRNA and control shRNA in the presence or absence of 3-MA (10 mM) treatment for 1 h and then starved by HBSS for 3 h. After 1-h incubating with ¹³¹I, the uptake of ¹³¹I in indicated cells was detected by a gamma counter (n = 3, *P < 0.01, **P > 0.01); (f) FTC-133/TPC-1 cells transfected with HMGB1 shRNA and control shRNA were starved by HBSS for 3 h and then treated with rapamycin (1 μM) for 12 h. After 1-h incubating with ¹³¹I, the uptake of ¹³¹I in indicated cells was detected by a gamma counter. Rap, rapamycin. (n = 3, *P < 0.01, **P > 0.01)

Fig. 4

ROS was sufficient for inducing HMGB1 translocation and enhancing autophagy (a) FTC-133/TPC-1 cells were pretreated with the antioxidant (NAC, 2 mM) for 1 h and then starved by HBSS for 3 h. ROS production was assessed by measuring the fluorescent intensity of DCF on a fluorescent plate reader. Incremental production of ROS was expressed as a percentage of control (n = 3, *P < 0.01, **P < 0.01). UT, untreated group; (b) Antioxidant and SOD1 RNAi regulated starvation-induced autophagy as measured by LC3-II expression. FTC-133/TPC-1 cells were pretreated with NAC (2 mM) for 1 h or SOD1 RNAi for 48 h, and then starved by HBSS for 3 h. LC3-I/II level was assayed by Western blot. (c) Antioxidant and SOD1 RNAi regulated starvation-induced HMGB1 translocation. FTC-133/TPC-1 cells were pretreated with NAC (2 mM) for 1 h or SOD1 RNAi for 48 h, and then starved by HBSS for 3 h. And the expression of nuclear/cytosolic HMGB1 was assayed by Western blot. Fibrillarin was a nuclear fraction control and tubulin a cytoplasmic fraction control

Fig. 5

ROS/AMPK/mTOR pathway was required for HMGB1-mediated autophagy regulating NIS expression (a) FTC-133/TPC-1 cells transfected with HMGB1 shRNA and control shRNA transfection were starved by HBSS for 3 h and then treated with rapamycin (1 μM) for 12 h. ROS production was assessed by measuring the fluorescent intensity of DCF on a fluorescent plate reader. Incremental production of ROS was expressed as a percentage of control. Rap, rapamycin. (n = 3, *P < 0.01, **P > 0.01); (b) FTC-133/TPC-1 cells transfected with HMGB1 shRNA and control shRNA were starved by HBSS for 3 h and then treated with rapamycin (1 μM) for 12 h. Flow cytometry was performed for measuring the ROS level by a DCFH-DA probe in indicated cells. Rap, rapamycin. (n = 3, *P < 0.01, **P > 0.01); (c) FTC-133/TPC-1 cells were transfected with HMGB1 shRNA and control shRNA and then starved by HBSS for 3 h. ATP levels were detected by ATP Assay Kit (n = 3, *P > 0.01); (d) FTC-133/TPC-1 cells were pretreated with NAC (2 mM) and then starved by HBSS for 3 h. LC3-I/II, NIS, p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6K and p70S6K levels were assayed by Western blot; (e) FTC-133/TPC-1 cells transfected with HMGB1 shRNA and control shRNA were starved by HBSS for 3 h and then treated with rapamycin (1 μM) for 12 h. LC3-I/II, NIS, p-mTOR and mTOR levels were assayed by Western blot. Rap, rapamycin

Fig. 6

HMGB1-mediated autophagy regulates the imaging of ^99mTcO4⁻ and NIS expression in tumor-bearing nude mice in vivo (a) Nude mice received a subcutaneous injection of HMGB1 shRNA and control shRNA cells and then food was withdrawn at 18 h before experimentation when murine tumor was > 150 mm³. Wholebody scintigraph of bearing-tumor mice was performed at 10 min after a tail injection of ^99mTc-pertechnetate. 1: thyroid gland, 2: transplanted tumor, 3: stomach. Arrows indicated that transplanted tumor was not visualized; (b) Biodistribution of ^99mTcO4 ^- in tumor-bearing nude mice (n = 3, *P < 0.01 vs. HMGB1 shRNA hunger group or control shRNA vehicle group); (c) Immunohistochemical staining of NIS was performed with isolated tumor at the end of experiment