TSH overcomes Braf(V600E)-induced senescence to promote tumor progression via downregulation of p53 expression in papillary thyroid cancer

Abstract

The BRAF(V600E) mutation is found in approximately 40% of papillary thyroid cancers (PTC). Mice with thyroid-specific expression of Braf(V600E) (TPO-Braf(V600E)) develop PTC rapidly with high levels of serum thyroid-stimulating hormone (TSH). It is unclear to what extent the elevated TSH contributes to tumor progression. To investigate the progression of Braf(V600E)-induced PTC (BVE-PTC) under normal TSH, we transplanted BVE-PTC tumors subcutaneously into nude and TPO-Braf(WT) mice. Regression of the transplanted tumors was observed in both nude and TPO-Braf(WT) mice. They were surrounded by heavy lymphocyte infiltration and oncogene-induced senescence (OIS) was demonstrated by strong β-gal staining and absence of Ki-67 expression. In contrast, BVE-PTC transplants continued to grow when transplanted into TPO-Braf(V600E) mice. The expression of Trp53 was increased in tumor transplants undergoing OIS. Trp53 inactivation reversed OIS and enabled tumor transplants to grow in nude mice with characteristic cell morphology of anaplastic thyroid cancer (ATC). PTC-to-ATC transformation was also observed in primary BVE-PTC tumors. ATC cells derived from Trp53 knockout tumors had increased PI3K/AKT signaling and became resistant to Braf(V600E) inhibitor PLX4720, which could be overcome by combined treatment of PI3K inhibitor LY294002 and PLX4720. In conclusion, BVE-PTC progression could be contained via p53-dependent OIS and TSH is a major disruptor of this balance. Simultaneous targeting of both MAPK and PI3K/AKT pathways offer a better therapeutic outcome against ATC. The current study reinforces the importance of rigorous control of serum TSH in PTC patients.

Figure 1.

Regression of BVE–PTC transplants in nude and TPO–Braf^WT mice. (A) Serum TSH level from TPO–Braf^V600E, TPO–Braf^WT and nude mice. The serum TSH levels from five TPO–Braf^V600E mice (5 months old) are all above the detection limit of 50 000 pg/ml. The average TSH levels from five TPO–Braf^WT and five nude mice of same age are 439.6 ± 39.8 and 426.4 ± 9.6 pg/ml, respectively. (B) Genotyping of TPO–Braf^V600E (a) and TPO–Braf^V600E–Trp53^−/− mice (b). The PCR product was run on a 1.5% agarose gel. The LSL–Braf^V600E was recombined only in the thyroid (activation of Braf^V600E) as a result of Cre-mediated deletion of a floxed transcriptional stop sequence. The LSL-Braf^V600E allele was not detected in the BVE-PTC cell line, indicating complete Cre-mediated recombination in the thyroid tumor cell line (a, lower panel), and no fibroblast contamination. Wild-type and mutant Trp53 alleles from BVE–PTC tumors are shown by 680-bp and 320-bp fragments, respectively. (C) Histology of BVE–PTC transplants. Tumors from 4- to 6-month-old TPO–Braf^V600E mice were transplanted subcutaneously into nude and TPO–Braf^WT mice. They were removed for histology after 4–7 months. The tumor transplants are surrounded by heavy lymphocyte infiltration (indicated by dark arrows) from both nude mice (a and b) and TPO–Braf^WT mice (c and d). Macrophages are often present in the empty spaces of tumor tissue (indicated by light arrows). (D) Tumor growth in TPO–Braf^V600E mice. TPO–Braf^V600E mice are about the half size of TPO–Braf^WT mice due to severe hypothyroidism and tumor transplants are indicated by arrows (a). The size of tumor transplants after 4-month implantation in TPO–Braf^V600E and TPO–Braf^WT mice (b). Histology of tumor transplants from TPO–Braf^V600E mice (c and d). There is more tumor tissue and less lymphocyte infiltration. Macrophages in the empty spaces of tumor tissue are indicated by light arrows.

Figure 2.

Braf^V600E-induced senescence in BVE–PTC transplants. (A) Western blot analysis of p53 expression from a 6-month-old BVE–PTC tumor transplant and primary BVE–PTC tumors of different ages. The expression of p53 is significantly higher in the tumor transplants. There is also a gradual increase in p53 expression from primary BVE–PTC tumors: lower expression in early tumors and higher expression in late tumors. Quantification of the western blot was performed using ImageJ software. (B) Strong SA-beta-Gal staining (blue color, a) and no Ki-67 immunostaining (c) in the BVE–PTC tumor transplants. No SA-beta-Gal staining (b) and strong Ki-67 immunostaining (d) in a primary BVE–PTC tumor. (C) Increased release of cytokines and chemokines in the fluid of BVE–PTC transplants. Cytokine and chemokine levels were assayed in the fluid of cysts from two BVE–PTC transplants and compared with those in the 48-h culture medium from a BVE–PTC cell line. The value from culture medium alone was subtracted and data are expressed as mean ± s.e.m. of triplicate wells. Only cytokines/chemokines with significant difference are shown (P < 0.05). (D) Decreased level of cytokines and chemokines in the fluid of cysts from BVE–PTC transplants. Only cytokines/chemokines with significant difference are shown.

Figure 3.

Inhibition of Braf^V600E-induced senescence by TSH in BVE–PTC cells. (A) Primary BVE–PTC cells were cultured in the presence of 2 mU/ml of bovine TSH for 2 months and stained for SA-beta-Gal activity (a) and p53 expression (c). Without bovine TSH in the culture, primary BVE–PTC cells could not replicate and was under senescence with strong SA-beta-Gal staining (b) and p53 immunostaining (d). (B) Western blot analysis of p53, p-AKT and E-Cadherin expression in a BVE–PTC cell line cultured for 72 h in the presence or absence of 10 mU/ml of bovine TSH. TSH can downregulate both p53 and E-Cadherin expression and increase p-AKT expression. (C) Quantification of p53 expression from the western blot was performed using ImageJ software. (D) Quantification of p-AKT expression from the western blot using ImageJ software.

Figure 4.

Trp53 knockout leads to ATC transformation and loss of Braf^V600E-induced senescence. (A) Transformation of PTC to ATC from a 5-month-old TPO–Braf^V600E–Trp53^+/− mouse (heterozygous Trp53 knockout, a, b, c) and a 3-month-old TPO–Braf^V600E–Trp53^−/− mouse (homozygous Trp53 knockout, d, e, f). ATC tumors (indicated by arrows) can be seen together with PTC. ATC tumors are larger from TPO–Braf^V600E–Trp53^−/− mice (d, e, f ) than from TPO–Braf^V600E–Trp53^+/− mice (a, b, c), and composed of highly pleomorphic giant cells and spindle cells with large, bizarre nuclei containing prominent nucleoli and mitotic figures (c and f). The papillary architecture is replaced by undifferentiated architecture. (B) Loss of tumor regression as a result of Trp53 knockout. Thyroid tumors were removed from 4-month-old TPO–Braf^V600E (with wild-type Trp53) mice and TPO–Braf^V600E–Trp53^−/− (with Trp53 knockout) mice. They were transplanted to nude mice and observed for 4 months. Regression of tumor transplant from a TPO–Braf^V600E mouse (left) and continued tumor growth from a TPO–Braf^V600E–Trp53^−/− mouse (right) are shown. (C) Histology of BVE–PTC tumor transplants. PTC architecture from tumor transplants with Trp53^−/− is completely replaced by undifferentiated cellular structure with many giant and spindle cells characteristic of ATC. Lymphocyte infiltration is not present (a and b). BVE–PTC tumor transplants with Trp53^+/+ are surrounded by heavy lymphocyte infiltration as indicated by arrows and are localized (c and d). (D) Western blot analysis of p53, p-ERK and p-AKT expression among normal thyroid, BVE–PTC, BVE–ATC-c1 and BVE–ATC-c2 cell lines. p-AKT expression is significantly increased in BVE–ATC-c1 and BVE–ATC-c2 cell lines as a result of Trp53 knockout.

Figure 5.

Effect of simultaneous inhibition of both MAPK and PI3K/AKT pathways against ATC cells. (A) Cell proliferation of BVE–PTC and BVE–ATC-c1 cell lines. The cells were plated into 96-well plates (1 × 10³ cells/well) and treated with PI3K inhibitor LY294002 or Braf^V600E inhibitor PLX4720 alone or in combination for up to 72 h. Cell proliferation was determined by a non-radioactive MTS assay kit. Cells treated with 0.1% dimethyl sulfoxide (DMSO) only were served as a vehicle control. Data are expressed as percentage of the vehicle control (100%) in mean ± s.e.m. of triplicate wells. (B) Colony formation assay. Cells were plated into 12-well plates (5 × 10² cells/well) and cultured in the presence of different concentrations of PLX4720, LY294002 or both for 14 days to determine long-term effects of PLX4720 and LY294002. Cells were then stained with 0.5% crystal violet dye (in methanol:de-ionized water, 1:5) for 10 min. After three washes with de-ionized water, the crystal violet dye was released from cells by incubation with 1% sodium dodecyl sulfate (SDS) for 2 h before optical density (OD)_{570 nm} measurement. Data are expressed as percentage of the vehicle control.