Abstract 1781: G1 phase melanoma cells escape proteasome inhibitor cytotoxicity

Abstract

Abstract Utilising the fluorescent ubiquitination-based cell cycle indicator (FUCCI), which facilitates real-time cell cycle tracking, we have demonstrated in vitro and in vivo that melanomas are composed of differentially cycling tumour cells in a subcompartment-specific distribution. Further, we have shown that targeting the endoplasmic reticulum 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 to investigate the effect of ER stress-inducing agents on the dynamics of cell division and cell death of individual melanoma cells within the complex tumor microenvironment, and to develop combination strategies that increase the efficacy of ER stress-inducing agents for melanoma therapy. FUCCI-melanoma cells were grown as 3D spheroids and implanted into a collagen matrix to mimic tumor architecture and microenvironment. Utilising the F-XBP1ΔDBD-venus reporter construct, which labels the cytoplasm in response to ER stress, we found that bortezomib induced ER stress, delayed cell cycle progression, and combination with fenretinide increased cell death in 2D and 3D culture. Flow cytometry and confocal microscopy indicated that treatment of FUCCI-melanoma cells with bortezomib induced G2 accumulation in 2D and 3D culture over the course of 24 h. In contrast, by 72 h the majority of cells were in G1 phase. Analysis of 2D and 3D real-time cell cycle imaging movies revealed that bortezomib induced both G1- and G2 arrest, but preferentially killed G2-phase cells. Consequently, pretreatment with temozolomide or fenretinide, leading to G2-arrest, sensitised melanoma cells to bortezomib cytotoxicity. In contrast, pretreatment with MEK inhibitors, leading to G1 arrest, inhibited bortezomib cytotoxicity in all melanoma cells, as did selective BRAF inhibitors in BRAF mutant melanoma cells. Our data suggest that bortezomib combined with fenretinide or temozolamide is a strategy worth exploring for the treatment of BRAF-inhibitor insensitive or resistant melanoma. Importantly, melanoma cells arrested in G1 are protected from bortezomib cytotoxicity, which excludes MAPK pathway inhibitors as combination partners. Citation Format: Sheena M. Daignault, David S. Hill, Kimberley A. Beaumont, Andrea Anfosso, Penny E. Lovat, Wolfgang Weninger, Nikolas K. Haass. G1 phase melanoma cells escape proteasome inhibitor cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1781. doi:10.1158/1538-7445.AM2015-1781