Targeting BRAFV600E with PLX4720 Displays Potent Antimigratory and Anti-invasive Activity in Preclinical Models of Human Thyroid Cancer

Abstract

Purpose B-RafV600E 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-RafV600E inhibitor, PLX4720, on proliferation, migration, and invasion both in human thyroid cancer cell lines (8505cB-RafV600E and TPC-1RET/PTC-1 and wild-type B-Raf) and in primary human normal thyroid (NT) follicular cells engineered with or without B-RafV600E. 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-RafV600E (heterozygous wild-type B-Raf/B-RafV600E) 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-RafV600E thyroid cancer cells and tumor aggressiveness. Normal thyroid cells were generated to be heterozygous for wild-type B-Raf/B-RafV600E, 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-RafV600E ATC.

p53 constrains progression to anaplastic thyroid carcinoma in a Braf -mutant mouse model of papillary thyroid cancer

“…Previous studies using MAPK pathway inhibitors in ATC had relied upon highly passaged human ATC cells in culture or transplanted into immunocompromised mice (16,31,32). Our results demonstrating an incomplete response of mATCs to PLX4720 are consistent with the reduced sensitivity of a variety of human thyroid carcinoma cell lines to MAPK pathway inhibition in cultured conditions.…”

A Potential Role for Immunotherapy in Thyroid Cancer by Enhancing NY-ESO-1 Cancer Antigen Expression

“…Total lysates were centrifuged at 15,000 g for 15 minutes at 4°C and supernatants collected and quantified using a Bradford protein assay kit (Pierce). Western blot analysis was performed using a standard procedure as described earlier (31), and the proteins were identified using specific antibodies.…”

RET/PTC rearrangement in benign and malignant thyroid diseases: a clinical standpoint

“…The discovery of genetic mutations involved in the pathogenesis of thyroid tumors has provided the basis for further improvements of the pre-surgical diagnostic methods. The most extensively studied mutation in this diagnostic context is BRAF V600E (67). Because this highly specific PTC marker is expressed only in about half of PTCs, more cancer markers are needed.…”