Targeting BRAFV600E with PLX4720 displays potent antimigratory and anti-invasive activity in preclinical models of human thyroid cancer

Abstract

Purpose: B-Raf(V600E) may play a role in the progression from papillary thyroid cancer to anaplastic thyroid cancer (ATC). We tested the effects of a highly selective B-Raf(V600E) inhibitor, PLX4720, on proliferation, migration, and invasion both in human thyroid cancer cell lines (8505c(B-RafV600E) and TPC-1(RET/PTC-1 and wild-type B-Raf)) and in primary human normal thyroid (NT) follicular cells engineered with or without B-Raf(V600E).

Experimental design: Large-scale genotyping analysis by mass spectrometry was performed in order to analyze >900 gene mutations. Cell proliferation and migration/invasion were performed upon PLX4720 treatment in 8505c, TPC-1, and NT cells. Orthotopic implantation of either 8505c or TPC-1 cells into the thyroid of severe combined immunodeficient mice was performed. Gene validations were performed by quantitative polymerase chain reaction and immunohistochemistry.

Results: We found that PLX4720 reduced in vitro cell proliferation and migration and invasion of 8505c cells, causing early downregulation of genes involved in tumor progression. PLX4720-treated NT cells overexpressing B-Raf(V600E) (heterozygous wild-type B-Raf/B-Raf(V600E)) showed significantly lower cell proliferation, migration, and invasion. PLX4720 treatment did not block cell invasion in TPC-1 cells with wild-type B-Raf, which showed very low and delayed in vivo tumor growth. In vivo, PLX4720 treatment of 8505c orthotopic thyroid tumors inhibited tumor aggressiveness and significantly upregulated the thyroid differentiation markers thyroid transcription factor 1 and paired box gene 8.

Conclusions: Here, we have shown that PLX4720 preferentially inhibits migration and invasion of B-Raf(V600E) thyroid cancer cells and tumor aggressiveness. Normal thyroid cells were generated to be heterozygous for wild-type B-Raf/B-Raf(V600E), mimicking the condition found in most human thyroid cancers. PLX4720 was effective in reducing cell proliferation, migration, and invasion in this heterozygous model. PLX4720 therapy should be tested and considered for a phase I study for the treatment of patients with B-Raf(V600E) ATC.

Figure 1.

p-ERK-1/ERK-2 expression in thyroid cancer cells. (A): B-Raf^V600E mutation in 8505c cells; TPC-1 cells have wt B-Raf and harbor the PIK3CA H1047R mutation and RET/PTC-1 translocation. (B): One hour of 1 μM PLX4720 treatment resulted in lower p-ERK-1/ERK-2 levels (by Western blotting) in 8505c cells, but higher levels in TPC-1 cells. p-ERK-1/ERK-2 levels were lower in 8505c cells and TPC-1 cells with 10 μM PLX4720. (C): 72 hours of PLX4720 treatment resulted in lower p-ERK-1/ERK-2 levels in 8505c cells and higher levels in TPC-1 cells. Abbreviations: DMSO, dimethyl sulfoxide; ERK, extracellular signal–related kinase; p, phosphorylated; wt, wild-type.

Figure 2.

PLX4720 inhibits proliferation in thyroid cancer cells. (A–C): 1 μM PLX4720 reduced BrdU incorporation in TPC-1 cells (*p = .01); 10 μM PLX4720 reduced BrdU uptake in 8505c and TPC-1 cells (***p < .001). (D): 1 μM PLX4720 significantly reduced S-phase (*p = .01) and caused G₁ phase arrest (**p < .01) in TPC-1 cells and 10 μM PLX4720 reduced S-phase (***p < .001) and caused G₁ phase arrest (**p < .001) in both TPC-1 and 8505c cells. Abbreviations: BrdU, bromodeoxyuridine; DMSO, dimethyl sulfoxide; wt, wild-type.

Figure 3.

Migration and invasion assays with PLX4720 treatment in thyroid cancer cell models. Treatment with 1 μM (A) or 10 μM (B) PLX4720 reduced migration and invasion in 8505c cells (***p < .001). (B): Migration of TPC-1 cells was lower with 10 μM PLX4720 (***p < .001). (C): PLX4720 inhibited p-ERK-1/ERK-2 in B-Raf^V600E-overexpressed primary human NT follicular cells, compared with NT cells transduced with the EV. (D): Migration and invasion of NT cells with B-Raf^V600E overexpression were inhibited by PLX4720 (***p < .001). Abbreviations: ERK, extracellular signal–related kinase; EV, empty vector; NT, normal thyroid; p, phosphorylated; wt, wild-type.

Figure 4.

Tumor growth and lung metastasis in an orthotopic mouse model of human anaplastic thyroid cancer. (A): Three weeks of PLX4720 treatment resulted in lower orthotopic tumor growth than in controls (***p < .001). Gross photo (B1) and 3D CT scan (B3) show a large B-Raf^V600E 8505c orthotopic thyroid carcinoma (control, arrow) and a dramatically smaller (B2 and B4) orthotopic tumor size with PLX4720 treatment (gross photo, arrow; 3D CT scans, asterisk) in SCID mice. (B5): Lung metastases (arrows) by GFP in control B-Raf^V600E 8505c orthotopic thyroid carcinoma SCID mice. (B6): Significantly fewer lung metastases in PLX4720-treated mice. (C) Control mice became cachectic between 28 and 35 days and lost an average of 23% of their baseline weight, whereas PLX4720-treated mice maintained their baseline weight (***p < .001). (D): Low proliferative index (percentage of Ki-67 protein nuclear expression) in PLX4720-treated B-Raf^V600E 8505c orthotopic tumors, compared with controls (**p < .01). Abbreviations: 3D, three-dimensional; CT, computed tomography; DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; SCID, severe combined immunodeficient.

Figure 5.

PLX4720 inhibits B-Raf^V600E-positive thyroid tumor aggressiveness in vivo. (A): Control mice with B-Raf^V600E 8505c orthotopic tumors (H&E stain) were large (A1) and showed extrathyroidal extension into the trachea (A1, A2) and skeletal muscle (A3) with atypical mitoses (A4, arrow); PLX4720-treated tumors (H&E stain) were small and discrete (A5–A7) (arrows) and largely confined to the thyroid bed, with atypical pyknotic nuclei (A8). (B): Control SCID mice (H&E stain) had metastatic foci of carcinoma to the lung interstitium (arrows, B1, B2) as well as a paratracheal lymph node (asterisk, (B3); higher power, arrow (B4)); PLX4720-treated mice had no evidence of metastasis to the lung (B5, B6) or lymph nodes (B7, B8). (C): PLX4720-treated tumors had greater nuclear staining for TTF-1 and PAX-8 (right) than untreated control mice (left). Abbreviations: H&E, hematoxylin and eosin; SCID, severe combined immunodeficient; TTF-1, thyroid transcription factor.

Figure 6.

Gene expression with PLX4720 treatment. Quantitative real-time RT-PCR shows mRNA copy number of CCND1 (A), TSP-1 (B), ITGA6 (C), and TTF-1 (D) mRNA in 8505c and TPC-1 cells after 24 hours or 72 hours of treatment with PLX4720 (either 1 μM or 10 μM). Abbreviations: CCND1, cyclin D1; ITGA6, integrin α6; RT-PCR, reverse transcriptase polymerase chain reaction; TSP-1, thrombospondin-1; TTF-1, thyroid transcription factor; wt, wild-type.