Synergistic antitumour activity of RAF265 and ZSTK474 on human TT medullary thyroid cancer cells

Abstract

Medullary thyroid cancer (MTC) is an aggressive malignancy responsible for up to 14% of all thyroid cancer-related deaths. It is characterized by point mutations in the rearranged during transfection (RET) proto-oncogene. The activated RET kinase is known to signal via extracellular signal regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K), leading to enhanced proliferation and resistance to apoptosis. In the present work, we have investigated the effect of two serine/threonine-protein kinase B-Raf (BRAF) inhibitors (RAF265 and SB590885), and a PI3K inhibitor (ZSTK474), on RET-mediated signalling and proliferation in a MTC cell line (TT cells) harbouring the RETC634W activating mutation. The effects of the inhibitors on VEGFR2, PI3K/Akt and mitogen-activated protein kinases signalling pathways, cell cycle, apoptosis and calcitonin production were also investigated. Only the RAF265+ ZSTK474 combination synergistically reduced the viability of treated cells. We observed a strong decrease in phosphorylated VEGFR2 for RAF265+ ZSTK474 and a signal reduction in activated Akt for ZSTK474. The activated ERK signal also decreased after RAF265 and RAF265+ ZSTK474 treatments. Alone and in combination with ZSTK474, RAF265 induced a sustained increase in necrosis. Only RAF265, alone and combined with ZSTK474, prompted a significant drop in calcitonin production. Combination therapy using RAF265 and ZSTK47 proved effective in MTC, demonstrating a cytotoxic effect. As the two inhibitors have been successfully tested individually in clinical trials on other human cancers, our preclinical data support the feasibility of their combined use in aggressive MTC.

Novel targeted therapeutics for MEN2

“…It is possible that VEGFR2 targeting contributed to in vivo tumor growth inhibition, similarly to all other effective RET inhibitors. More recently, another group explored synergistic inhibition of RET-mediated signaling and cell growth by the combination of RAF265 with a different PI3K inhibitor, named ZSTK474 (Bertazza et al 2015). ERK and AKT activation, cell growth and survival, and calcitonin expression and secretion were all significantly better inhibited by the combination compared to single treatments, in TT cells.…”

BRAFwild papillary thyroid carcinoma has two distinct mRNA expression patterns with different clinical behaviors

“…Several groups have reported the action of RAF inhibitor against VEGFR2 in thyroid cancer. 30,31 Interestingly, in this study, BRAF wild PTCs in cluster 2 showed higher VEGFR2 protein than BRAF V600E PTCs (see Figure 5). Subsequently, we tried to select genes that were differentially expressed between PTCs in cluster 1 and BRAF wild PTCs in cluster 2 (see Figure 6).…”

“…In the RPPA analysis, the VEGFR2 protein expression in BRAF wild PTCs of cluster 2 was higher than that in BRAF wild PTCs of cluster 1 (see Figure ). Several groups have reported the action of RAF inhibitor against VEGFR2 in thyroid cancer . Interestingly, in this study, BRAF wild PTCs in cluster 2 showed higher VEGFR2 protein than BRAF V600E PTCs (see Figure ).…”

Combinatorial Therapies in Thyroid Cancer: An Overview of Preclinical and Clinical Progresses

“…With the capacity of the molecule to target RET mutation, the potential of RAF265 to effectively treat the MTC form is important. In this context, combined with the PI3K inhibitor ZSTK474, RAF265 induced a strong cytotoxic effect and a strong inhibition of VEGFR-2 phosphorylation in TT cells harboring genetic alterations in RET [72].…”