Induction of sodium/iodide symporter (NIS) expression and radioiodine uptake in non-thyroid cancer cells

Abstract

Background: This study was designed to explore the therapeutic potential of suppressing MAP kinase and PI3K/Akt pathways and histone deacetylase (HDAC) to induce the expression of sodium/iodide symporter (NIS) and radioiodine uptake in non-thyroid cancer cells.

Methods: We tested the effects of the MEK inhibitor RDEA119, the Akt inhibitor perifosine, and the HDAC inhibitor SAHA on NIS expression in thirteen human cancer cell lines derived from melanoma, hepatic carcinoma, gastric carcinoma, colon carcinoma, breast carcinoma, and brain cancers. We also examined radioiodine uptake and histone acetylation at the NIS promoter in selected cells.

Results: Overall, the three inhibitors could induce NIS expression, to various extents, in melanoma and all the epithelial carcinoma-derived cells but not in brain cancer-derived cells. SAHA was most effective and its effect could be significantly enhanced by RDEA119 and perifosine. The expression of NIS, at both mRNA and protein levels, was most robust in the melanoma cell M14, hepatic carcinoma cell HepG2, and the gastric carcinoma cell MKN-7 cell. Radioiodine uptake was correspondingly induced, accompanied by robust increase in histone acetylation at the NIS promoter, in these cells when treated with the three inhibitors.

Conclusions: This is the first demonstration that simultaneously suppressing the MAP kinase and PI3K/Akt pathways and HDAC could induce robust NIS expression and radioiodine uptake in certain non-thyroid human cancer cells, providing novel therapeutic implications for adjunct radioiodine treatment of these cancers.

Figure 1. Target effects of inhibitors of the MAP kinase and PI3K/Akt pathways and HDAC in various non-thyroid human cancer cells.

The MEK inhibitor RDEA119, the Akt inhibitor perifosine, and the HDAC inhibitor SAHA were used to respectively target these signaling pathways or molecules. Cells were treated for 30 hrs with 0.5 µM RDEA119, 5 µM perifosine or 0.5 µM SAHA as indicated. DMSO or PBS was used in parallel as the vehicle control. Cells were lysed for Western blotting after treatments to reveal the levels of phosphorylated ERK (p-ERK), phosphorylated Akt (p-Akt), and acetylated histone (Ac-His) with specific antibodies. “+”, treatment with the indicated inhibitor; “−”, treatment with vehicle.

Figure 2. Western blotting analysis of NIS protein expression in M14, HepG2 and MKN-7 cells after treatment with the MEK, Akt and HDAC inhibitors.

Cells were treated for 30 hrs with combination use of RDEA119, perifosine, and SAHA at the concentrations described in Fig. 1, followed by standard Western blotting analysis of cell lysates using the specific primary antibody against NIS. The expression level of ß-actin was analyzed in parallel for quality control. “Control”, treatment of cells with vehicle; “R+P+S”, treatment of cells with combined use of RDEA119, perifosine and SAHA.

Figure 3. Induction of radioiodine uptake in M14, HepG2 and MKN-7 cells by treatment with the MEK, Akt and HDAC inhibitors.

Cells were treated with RDEA119, perifosine and SAHA as described in Fig. 2, followed by incubation with Na¹²⁵I for 1 hr. Parallel cells were additionally treated with the NIS blocker NaClO₄ to obtain non-specific radioiodine uptake/binding with the cells. Cells were then washed, lysed, and measured for radioactivity. Cell radioiodine uptake is presented as cpm/10⁶ cells (on the y-axis of the figure) after correction for the non-specific radioiodine binding. Detailed experimental procedures are as described in the Materials and Methods. “Control”, treatment of cells with vehicle; “R+P+S”, treatment of cells with combination use of RDEA119, perifosine and SAHA. ** In comparison with control, p<0.01.

Figure 4. Effects of the treatment with MEK, Akt and HDAC inhibitors on histone acetylation at the NIS promoter in M14, HepG2 and MKN-7 cells.

Cells were treated with SAHA alone or SAHA in combination with RDEA119 and perifosine as described in Fig. 2. Histone acetylation levels at the three regions (P1, P2, and P3) that comprise the minimal essential promoter of the NIS gene were analyzed by chromatin immunoprecipitation (ChIP) analysis as described in Materials and Methods. Acetylation status of both histone H3 and H4 was examined using specific antibodies for ChIP. Non-specific IgG antibodies were used as control. The results for M14, HepG2 and MKN-7 cells are presented in Figs. 4a, 4b and 4c, respectively. For each cell, the upper panel shows the actual results of PCR on the DNA fragments obtained by ChIP using non-specific control IgG, anti-acetylated H3 (Anti Ac-H3) antibody or anti-acetylated H4 (Anti Ac-H4) antibody. Identical amounts of pre-immunoprecipitation cell lysates from different treatment conditions were used to start the immunoprecipitation. An aliquot of pre-immunoprecipitation cell lysate was directly used to isolate DNA as “Input” control. The PCR results reflect the histone acetylation levels. Presented in the lower panel for each cell is a bar graph showing quantitatively the acetylation levels of H3 and H4 at the three regions of NIS promoter based on densitometric measurements of the upper panel. The results are normalized by dividing the corresponding input signals and are presented as the ratio of the indicated treatment over the control. “Control”, treatment of cells with vehicle; “SAHA”, treatment of cells with SAHA alone; “R+P+S”, treatment of cells with combined use of RDEA119, perifosine and SAHA.