We
have shown in vitro and in vivo, that melanomas are composed
of differentially cycling tumour cells in a subcompartment-specific distribution,
which may result in differential sensitivity to apoptosis. Further, we
have demonstrated that targeting the endoplasmic reticulum (ER) with
fenretinide (synthetic retinoid) or bortezomib (26S proteasome inhibitor)
induces cell cycle arrest and apoptosis of metastatic
melanoma cells in vitro and in vivo. This study aims (1) to
investigate the effect of ER
stress-inducing drugs on the dynamics of cell division and cell death
of individual melanoma cells within the complex tumour microenvironment, and
(2) to develop combination strategies that increase the efficacy of
ER stress-inducing agents for the treatment of melanoma.
We made use of the fluorescent ubiquitination-based cell cycle
indicator (FUCCI), which facilitates the identification of individual
proliferating cells in G1 versus S/G2/M cell cycle phases, in combination
with confocal and multiphoton microscopy to track melanoma cell
proliferation in realtime. We utilised the F-XBP1ΔDBD-venus reporter
construct, which labels the cytoplasm of cells in response to ER stress.
We show that bortezomib induced ER stress, delayed cell cycle progression,
and combination with fenretinide increased cell death in our 3D melanoma
model. While the selective BRAF-inhibitor vemurafenib induced G1
arrest, bortezomib induced G1and G2 arrest, but preferentially killed
G2-phase cells. Temozolomide enhanced the effect of bortezomib.
However, MEK inhibitors blocked the effect of bortezomib in all melanoma
cells, as did BRAF-inhibitors in BRAF mutant cells.
Our data suggest that bortezomib combined with fenretinide or temozolamide
is a therapeutic strategy worth exploring for the treatment
of BRAF-inhibitor insensitive or resistant melanoma. Importantly,
melanoma cells in G1 are protected from the cytotoxic effect of
bortezomib, which excludes MEK or BRAF inhibitors as combination partners.