The Sphingosine Kinase 2 Inhibitor ABC294640 Restores the Sensitivity of BRAFV600E Mutant Colon Cancer Cells to Vemurafenib by Reducing AKT-Mediated Expression of Nucleophosmin and Translationally-Controlled Tumour Protein.

Petra Grbčić,Thomas O Eichmann,Mirela Sedić,Sandra Kraljević Pavelić

doi:10.3390/ijms221910767

Abstract

Vemurafenib (PLX4032), small-molecule inhibitor of mutated BRAFV600E protein, has emerged as a potent anti-cancer agent against metastatic melanoma harboring BRAFV600E mutation. Unfortunately, the effect of PLX4032 in the treatment of metastatic BRAF mutated colorectal cancer (CRC) is less potent due to high incidence of fast-developing chemoresistance. It has been demonstrated that sphingolipids are important mediators of chemoresistance to various therapies in colon cancer. In this study, we will explore the role of major regulators of sphingolipid metabolism and signaling in the development of resistance to vemurafenib in BRAF mutant colon cancer cells. The obtained data revealed significantly increased expression levels of activated sphingosine kinases (SphK1 and SphK2) in resistant cells concomitant with increased abundance of sphingosine-1-phosphate (S1P) and its precursor sphingosine, which was accompanied by increased expression levels of the enzymes regulating the ceramide salvage pathway, namely ceramide synthases 2 and 6 and acid ceramidase, especially after the exposure to vemurafenib. Pharmacological inhibition of SphK1/SphK2 activities or modulation of ceramide metabolism by exogenous C6-ceramide enhanced the anti-proliferative effect of PLX4032 in resistant RKO cells in a synergistic manner. It is important to note that the inhibition of SphK2 by ABC294640 proved effective at restoring the sensitivity of resistant cells to vemurafenib at the largest number of combinations of sub-toxic drug concentrations with minimal cytotoxicity. Furthermore, the obtained findings revealed that enhanced anti-proliferative, anti-migratory, anti-clonogenic and pro-apoptotic effects of a combination treatment with ABC294640 and PLX4032 relative to either drug alone were accompanied by the inhibition of S1P-regulated AKT activity and concomitant abrogation of AKT-mediated cellular levels of nucleophosmin and translationally-controlled tumour protein. Collectively, our study suggests the possibility of using the combination of ABC294640 and PLX4032 as a novel therapeutic approach to combat vemurafenib resistance in BRAF mutant colon cancer, which warrants additional preclinical validation studies.

Highlights

Vemurafenib (PLX4032), a small-molecule inhibitor of mutated BRAFV600E protein, has emerged as a potent anti-cancer agent against metastatic melanoma harbouringBRAFV600E mutation that occurs in codon 600 resulting in the substitution of a valine for a glutamic acid (V600E) leading to a constitutive activation of the BRAF protein
We found significantly increased expression levels of activated sphingosine kinases 1 and 2 in resistant cells concomitant with increased abundance of sphingosine-1-phosphate (S1P) and its precursor sphingosine, which was accompanied by a marked increase in the expression levels of the enzymes regulating the ceramide salvage pathway, including ceramide synthases 2 and 6 and acid ceramidase, especially after the exposure to vemurafenib
Consistent with previous findings in literature [1,9], we confirmed elevated expression levels of phospho-c-RAF, phospho-ERK1/2, phospho-MEK1/2 and phospho-AKT in resistant cell lines under basal conditions and especially upon the treatment with PLX4032 (Supplementary Figure S2), which again demonstrated that activation of the RAF/MEK/extracellular signal-regulated kinase (ERK) and AKT signalling could be associated with the development of vemurafenib-resistant phenotype in colon cancer

Summary

Introduction

BRAFV600E mutation that occurs in codon 600 resulting in the substitution of a valine for a glutamic acid (V600E) leading to a constitutive activation of the BRAF protein. The latter promotes cell proliferation in the absence of necessary growth factors under normal conditions. It has been shown that BRAF mutant colon cancer cell lines have higher levels of phospho-protein kinase B (AKT) indicative of an activation of the phosphoinositide 3-kinase (PI3K)–AKT pathway when compared to BRAF mutant melanoma [1]. BRAF mutant colon cancer cell line HT-29, with acquired resistance to vemurafenib, displayed an increased expression of activated AKT. Many gaps remain in our current understanding of the acquired resistance to BRAF inhibition in BRAF mutant colorectal cancer that preclude successful management of the colorectal cancer patients carrying BRAF mutation

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Results

Conclusion