The Effect And Molecular Mechanism Of FoxP3 On NIS Expression In Papillary Thyroid Carcinoma

BackgroundPapillary thyroid carcinoma(PTC) accounts for the majority of thyroid cancers and generally carries an excellent prognosis, with a 10-year survival rate of 90%. Following thyroidectomy, radioiodine ablation is the primary treatment for PTC; however, some patients may fail to respond when the cancer has lost radioiodine avidity. Multiple lines of evidence suggest decreased expression of NIS(sodium iodine symporter) is associated with this loss of radioiodine responsiveness. NIS, is an intrinsic plasma membrane protein that mediates active transport of iodine in thyroid cells, where it is concentrated and organified. It is mainly regulated by TSH, which increases transcript and protein levels of NIS, but is also required for targeting of NIS to the thyrocyte plasma membrane. In addition to TSH, TGF-β1, estrogen and iodine also play an indispensable role in NIS modulation. TGF-β1, which inhibits thyroid function by antagonizing the biological effect of TSH, is also a potent immunosuppressor and its upregulation is associated with autoimmunity, inflammation, and cancer. The Smads are a family of intracellular signaling transducers that function downstream of the TGF-β1 signaling pathway. They are the only TGF-β1 receptor substrates with the ability to amplify signals. TGF-β1 binds to a specific kinase(TβR-II) on the cell surface, leading to activation of TβR-I, which then phosphorylates members of the Smad family(R-Smad2 and R-Smad3). The phosphorylated R-Smads form oligomers with Smad4 and immediately translocate to the nucleus, where the Smad compounds regulate the transcription of its target gene.Forkhead box P3(Fox P3), which belongs to the forkhead box family, is a key regulator of CD4+CD25+ regulatory T cells(Tregs) and is considered the only definitive marker of Tregs. Fox P3 was considered to be specific to Tregs until Hanz et al. identified it in pancreatic carcinoma. Since then, many studies have confirmed that it is also expressed in a variety of tumor cells and tumors that are Fox P3-positive usually have increased malignance which may be due to the suppression of immune function. Studies have shown that Fox P3 in cancer cells mimics its function in the Tregs, which inhibit the proliferation and function of tumor specific T cells through the secretion of immunosuppressive cytokines such as TGF-β1 and IL-10, thus leading to resistance of the tumors to immune destruction. More importantly, Fox P3 expression is significantly associated with resistance to radioiodine treatment.In light of these findings, we supposed there might be a relationship between Fox P3 and NIS. TGF-β1 is a potent inhibitor of NIS transcription in normal thyroid epithelial cells and Fox P3 in cancer cells induces the secretion of TGF-β1; thus, we hypothesized that Fox P3 may inhibit the expression and membrane targeting of NIS by inducing TGF-β1 secretion and subsequent activation of the Smad signaling pathway. In this study, 90 papillary thyroid carcinomas and 40 normal thyroid tissues were examined by immunohistochemistry and real-time PCR. A significant decrease of NIS expression was observed in the Fox P3-positive group when compared with the Fox P3-negative group.Importantly, through lentiviral vector–mediated overexpressing of constitutively active Fox P3 in TPC-1 cells, we investigate the effect and molecular mechanism of Fox P3 on the activation of TGF-β1/Smad pathway and expression of NIS. Objective1.To characterize the expression of Fox P3 in thyroid cancer cells and investigate whether it was associated with NIS repression.2.To study the effect and molecular mechanism of Fox P3 on NIS expression in TPC-1 cells. Methods1. Samples from 90 PTCs as well as 40 normal thyroid tissues were examined for Fox P3 and NIS by immunohistochemistry and real-time PCR.2. The human PTC-derived TPC-1 cell line was cultured in RP-1640 medium supplemented with 10% donor calf serum and a six-hormone mixture. The cells were pre-treated with human recombinant TGF-β1 for indicated time. Real-time PCR and Western blot were performed to analyze the expression levels of NIS.3. TPC-1 cells were transfected with constitutively active Fox P3 lentiviral vectors(LV-CA-Fox P3) or empty lentiviral vectors(LV-NC-GFP) which were constructed by our research group in previous study. Transfection efficiency was observed by inverted fluorescence microscopy and detected by flow cytometry.4. Cells were divided into the control group, LV-CA-Fox P3(LV-CA) group and LV-NC-GFP(LV-NC) group. Real-time PCR was conducted to examine the m RNA levels of Fox P3 and TGF-β1, Western blot was performed to analyze the protein levels of Fox P3, TGF-β1, TβR-I, Smad and p Smad3. Immunofluorescence assays were used to observe the plasma membrane localization of TβR-I and phosphorylation levels of Smad3. In addition, the induction of TGF-β1 secretion in cell culture supernatants by Fox P3 was analyzed by ELISA.5. we divided the cells into the control group, LV-CA group, LV-CA groups with a neutralizing TGF-β1 antibody of indicated concentrations(1, 5, 10 μg/m L) and LV-NC group. Real-time PCR and Western blot were performed to analyze the expression levels of NIS. Results1.IHC revealed that 74% the tumor specimens scored positive. In contrast, all normal cases were negative for Foxp3 staining. correlation of Fox P3 expression with clinicopathological features was significant only for the presence of extrathyroid invasion(P<0.05)and lymph node metastasis(P<0.05).2.NIS staining was located almost exclusively at the plasma membrane in normal thyroid tissues; however, staining was less intense in tumor tissues, particularly in those that were Fox P3-positive. Moreover, the limited amounts of NIS protein were confined almost exclusively to the cytoplasm rather than being targeted to the plasma membrane.3. The Fox P3-positive group had a lower rate of NIS-positive staining(31.8% vs. 66.7%). NIS transcript levels were also significantly reduced in tumor samples vs. normal tissue and in the Fox P3-positive group vs. the Fox P3-negative group.4.When the cells were treated with increasing concentrations of recombinant TGF-β1(1, 5, 10 ng/m L), m RNA and protein levels of NIS decreased and got a minimum at the concentration of 10 ng/ml.5. Inverted fluorescence microscopy showed that TPC-1 cells generated abundant GFP in the LV-CA and LV-NC group, but no GFP was present in the non-transfected group. Flow cytometry revealed that GFP positivity, equivalent to the transfection efficiency, was 0.2%(NC group), 99.3%(LV-CA group), and 99.6%(LV-NC group).6.After transfection, Fox P3 m RNA and protein levels increased significantly in the LVCA-Fox P3 group vs. the control(P<0.05). Meanwhile, there was a noticeable increase of TGF-β1 m RNA and protein expression in LVCA-Fox P3 cells vs. the control( P<0.05). ELISA showed that Fox P3 overexpression led to >5-fold upregulation of TGF-β1 protein levels in the medium. Western blotting revealed a noticeable increase in TβR-I and p Smad3 protein levels(P<0.05), but no difference in total Smad3 protein levels. Similarly, there was no significant difference between the control group and the LV-NC group for the whole targets.7.Immunofluorescence assays showed that Fox P3 was mainly expressed in the nucleus of the TPC-1 cells and there was no Fox P3 expression in the control group and LV-NC group. Immunofluorescence also confirmed that plasma membrane localization of TβR-I and phosphorylation of Smad3 were notably induced in the transfected TPC-1 cells.8.When compared with the NC group, the m RNA and protein levels of NIS decreased significantly(P<0.05). We also observed that Fox P3-induced repression of NIS targeting to the plasma membrane was partially abrogated(up to 71.2%) when cells were incubated with increasing amounts of TGF-β1 neutralizing antibody. Real-time PCR also showed that the decreased expression levels of NIS m RNA were similarly rescued by neutralizing TGF-β1 antibody. Conclusions1.Fox P3 was highly expressed in tumor cells of papillary thyroid carcinoma and was associated with extrathyroid invasion, lymph node metastasis and decreased NIS expression.2.Fox P3 could compromise NIS expression as well as its membrane targeting through the activation of TGF-β1/Smad pathway.