HER inhibitor promotes BRAF/MEK inhibitor-induced redifferentiation in papillary thyroid cancer harboring BRAFV600E

Abstract

Redifferentiation therapy with BRAF/MEK inhibitors to facilitate treatment with radioiodine represents a good choice for radioiodine-refractory differentiated thyroid carcinoma, but recent initial clinical outcomes were modest. MAPK rebound caused by BRAF/MEK inhibitors-induced activation of HER2/HER3 is a resistance mechanism, and combination with HER inhibitor to prevent MAPK rebound may sensitize BRAFV600E-mutant thyroid cancer cells to redifferentiation therapy. To evaluate if inhibiting both BRAF/MEK and HER can produce stronger redifferetiation effect, we tested the effects of BRAF/MEK inhibitor dabrafenib/selumetinib alone or in combination with HER inhibitor lapatinib on the expression and function of iodine- and glucose-handling genes in BRAFV600E-positive BCPAP and K1 cells, using BHP 2-7 cells harboring RET/PTC1 rearrangement as control. Herein, we showed that lapatinib prevented MAPK rebound and sensitized BRAFV600E-positive papillary thyroid cancer cells to BRAF/MEK inhibitors. Dabrafenib/selumetinib alone increased iodine-uptake and toxicity and suppressed glucose-metablism in BRAFV600E-positive papillary thyroid cancer cells. When lapatinib was added, more significant effects on iodine- and glucose-handling gene expression, cell membrane location of sodium/iodine symporter as well as radioiodine uptake and toxicity were observed. Thus, combined therapy using HER inhibitor and BRAF/MEK inhibitor presented more significant redifferentiation effect on papillary thyroid cancer cells harboring BRAFV600E than BRAF/MEK inhibitor alone. In vivo and clinical studies assessing such combined targeted redifferentiation strategy were warranted.

Keywords: dabrafenib; glucose; iodine; papillary thyroid cancer; redifferentiation.

Figure 1

(A) Western blot of lysates of BCPAP cells treated with 0.1 μM dabrafenib with or without 1 μM lapatinib for indicated times. Lapatinib blocked the dabrafenib-induced HER3 and AKT phosphorylation and the pERK1/2 rebound. (B) BCPAP cells were treated with 2.5 μM selumetib with or without 1 μM lapatinib and collected at 0, 1, 8, and 48 h post-treatment. Lysates were Western blotted for HER3 and ERK.

Figure 2

Effects of different treatment on the mRNA levels of sodium iodine symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), thyroid-stimulating hormone receptor (TSHR); and on glucose transporter isoforms (GLUT1 and GLUT3) genes in BCPAP cells, K1 cells and BHP 2-7 cells. Cells were treated with 0.1 μM dabrafenib/2.5 μM selumetinib and 1 μM lapatinib alone or in combination. Data are presented as means ± SD. *P < 0.05; **P < 0.01 for comparison with control. Con: control (DMSO); Da: dabrafenib; Se: selumetinib; La: lapatinib.

Figure 3

Western blot demonstrating the effects of different treatment on the protein levels of sodium iodine symporter (NIS), thyroglobulin (Tg), thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), glucose transporter-1 (GLUT1) in BCPAP (left) and K1 (right) cells. Cells were treated with 0.1 μM dabrafenib alone or in combination with 1 μM lapatinib. β-actin was used as positive control.

Figure 4

Immunofluorescent microscopic analysis of NIS protein expression in BCPAP cells. Cells were treated with 0.1 μM dabrafenib/2.5 μM selumetinib and 1 μM lapatinib alone or in combination. Double immunofluorescent microscopy was displayed with the blue color representing DAPI nuclear staining and the green color representing NIS staining. NIS staining was negative in the nontreated and lapatinib treated cells. In cells treated with dabrafenib/selumetinib, NIS staining was notable. The most robust expression of NIS was seen in the combined treatment groups. Da: dabrafenib; Se: selumetinib.

Figure 5

Radioactive iodine uptake in BCPAP cells, K1 cells, and BHP 2-7 cells. Cells were treated with 0.1 μM dabrafenib/ 2.5 μM selumetinib and 1 μM lapatinib alone or in combination. Data are expressed as mean ± SD of values. *P < 0.05, **P < 0.01 compared with untreated cells. ●P < 0.05, ● ●P < 0.01 compared with NaClO₄ treated cells. Da: dabrafenib; Se: selumetinib; La: lapatinib.

Figure 6:

In vitro clonogenic assay. Data are represented as number of colonies in BCPAP cells and K1 cells treated with DMSO, 0.1 μM dabrafenib/2.5 μM selumetinib alone or in combination with 1 μM lapatinib with (¹³¹I+) or without ¹³¹I (¹³¹I-). Data are expressed as mean ± SD. Da: dabrafenib; Se: selumetinib; La: lapatinib.