Autophagic flux blockage by accumulation of weakly basic tenovins leads to elimination of B-Raf mutant tumour cells that survive vemurafenib

Abstract

Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.

Fig 1. Structure activity relationship study of tenovin-6 analogues.

(A) Sulforhodamine B (SRB) assays and structures for indicated compounds illustrating the ability to reduce cell growth in ARN8 human melanoma cells or in human normal dermal fibroblast (HNDF) cells with treatment for 72 hours prior to staining with SRB. (B) Flow cytometric analysis of cell cycle progression for ARN8 human melanoma and HNDF cells treated with the indicated compounds for 48 hours prior to fixation and staining with propidium iodide and analysis by flow cytometry.

Fig 2. Induction of LC3B is correlated with the presence of an aliphatic tertiary amine.

(A) MDA-MB468 human breast cancer cells treated with indicated doses of the stated compounds or vehicle for six hours with LC3B and alpha-tubulin detected by western blot. See details of the presence or absence of the aliphatic tertiary amine in Table 1. (B) MDA-MB468 human breast cancer cells treated with indicated compounds at 10 μM for indicated times with LC3B, p62 and alpha-tubulin detected by western blot. (C) MDA-MB468 human breast cancer cells treated with indicated doses of the stated compounds with or without chloroquine for six hours with LC3B and alpha-tubulin detected by western blot.

Fig 3. Tenovins prevent autophagic flux by a similar mechanism to chloroquine.

(A) ARN8 cells stained with 500 nM of LysoTracker red after treatment with either 15 μM of tenovin-50-OH or tenovin-50, 100 μM chloroquine or vehicle for two hours prior to imaging at 32× magnification. (B) ARN8 cells treated with indicated compounds for six hours with LC3B and alpha-tubulin levels detected by western blot. (C) ARN8 and MDA-MB468 cells treated with 10 μM of tenovin-6, tenovin-39, tenovin-50 or 100 μM chloroquine (CQ) for six hours. Shown are representative images obtained using the ImageStream platform of LysoTracker stained cells and graphs of the mean fluorescence intensity from two independent biological repeats with each separate treatment group having >10 000 events in the singlet and focused gates counted for the analysis.

Fig 4. Tenovins eliminate melanoma cells in culture due to blockage of autophagic flux.

Clonogenic assay in ARN8 human melanoma cells showing the ability of indicated tenovins to eliminate tumor cells in culture. (i) Cells were treated for 72 hours and stained with Giemsa to show pre-recovery cell density. (ii) Cells were treated for 72 hours with the indicated compounds. Following treatment, the medium was replaced and the cells allowed to grow for a set period of time as described in materials and methods followed by staining with Giemsa solution to show colonies of proliferating cells during recovery in compound-free medium.

Fig 5. Autophagic flux blockage by tenovins leads to elimination of remaining melanoma cells following vemurafenib treatment.

(A) SRB assay indicating surviving populations of ARN8 melanoma cells upon treatment with indicated doses of vemurafenib. (B) Flow cytometric analysis of ARN8 cells treated for 73 hours with indicated dose of vemurafenib and stained with propidium iodide. (C) Clonogenic assay in ARN8 melanoma cells treated as indicated for (i) 72 hours followed by staining with Giemsa to show surviving cells or (ii) 72 hours followed by four days of recovery in compound-free medium followed by staining with Giemsa stain to show cells capable of proliferating. (D) Clonogenic assay using ARN8 melanoma cells treated with (i) vemurafenib at 10 μM dose for 72 hours followed by a 72 hour treatment with vemurafenib at 10 μM and tenovin-50 at indicated doses or (ii) vemurafenib at indicated doses for 72 hours followed by 72 hours treatment with tenovin-50 at 7.5 μM. Both treatment groups were allowed four days of recovery in compound-free medium followed by staining with Giemsa solution to show cells capable of proliferating.