Utilising the fluorescent ubiquitination-based cell cycle indicator (FUCCI), facilitating real-time cell cycle tracking, we demonstratethat melanomas are composed of differentially cycling tumour cells in a subcompartment-specific distribution.
This study investigates the effect of ER stress-inducing agents, fenretinide (synthetic retinoid) and bortezomib (26S proteasome inhibitor), on the dynamics of cell division and cell death of individual melanoma cells within tumor microenvironments to increase therapy efficacy.
FUCCI-melanoma cells were grown as 3D spheroids and embedded into a collagen matrix mimicing tumor microhabitats, or as xenografts in NOD/SCID mice. Utilising the F-XBP1ΔDBD-venus reporter construct that tags the cytoplasm in response to ER stress, we found that bortezomib induced ER stress and delayed cell cycle progression. Flow cytometry and confocal microscopy indicated that treatment of FUCCI-melanoma cells with bortezomib induced G2 accumulation in both cultures over 24 hours, and by 72 h were predominately in G1 phase. Additionally, heightened cell death in 2D and 3D culture was achieved with bortezomib in combination with fenretinide. Interestingly, bortezomib generated G1- and G2 arrest, but preferentially killed G2-phase cells. While temozolomide enhanced cytotoxic effects of bortezomib, MEK inhibitors blocked this effect in all melanoma cells, as did selective BRAF inhibitors in BRAF mutant cells.
Our findings suggest that bortezomib combined with fenretinide or temozolamide is promising for treatment of BRAF-inhibitor insensitive or resistant melanoma. Importantly, melanoma cells arrested in G1 are protected from bortezomib cytotoxicity, excluding MAPK pathway inhibitors as combination options.