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Characterization of verteporfin as an inhibitor of autophagosome formation and its therapeutic potential in cancer

University of British Columbia

Autophagy is a cellular “self-eating” process that enables cells to degrade and recycle cytoplasmic materials both as a housekeeping mechanism and in response to extracellular stress. Based on preclinical studies, autophagy promotes cell survival in the nutrient-deprived tumour environment and in response to several cancer therapy agents, making it a prospective therapeutic target. Due to a lack of pharmacologically suitable and selective autophagy inhibitors, a phenotypic automated fluorescence microscopy assay was designed and used to screen > 3,500 drugs and pharmacological agents for novel inhibitors of autophagosome accumulation. Verteporfin, a benzoporphyrin derivative used in photodynamic therapy, was the only active compound identified. As a photosensitizer, verteporfin generates large amounts of singlet oxygen upon light irradiation, which elicits a cell-death response. In the absence of light activation, verteporfin is nontoxic and inhibits starvation- and drug-induced autophagy. Biochemical and microscopy assays revealed that verteporfin prevents autophagic sequestration and degradation downstream of LC3 lipidation and membrane association. p62 is a scaffold protein that oligomerizes and links poly-ubiquitinated proteins to the autophagosome membrane by binding LC3, thus delivering both its cargo and itself for lysosomal degradation. Western blot analysis revealed that verteporfin produces SDS-stable high-MW p62, which is highly oxidized, and is likely a product of p62 crosslinking. The mechanism of high-MW p62 generation by verteporfin was discovered to be low-level singlet oxygen production, and the appearance of high-MW p62 correlated with autophagy inhibition. p62 co-immunoprecipitation experiments revealed that its association with EGFP-LC3 was not affected by verteporfin, but binding to poly-ubiquitinated cargo was disrupted. Therefore, non-photoactivated verteporfin generates low-level singlet oxygen that induces p62 oxidation and high-MW products that may interfere with autophagosome formation. Verteporfin was used to evaluate the therapeutic potential of autophagy inhibition using two different tumour xenograft models. Verteporfin did not show anti-tumour activity in a JIMT-1 breast cancer model, but it did enhance the anti-tumour and survival effects conferred by gemcitabine in a BxPC-3 pancreatic cancer model. The characterization of an early autophagy inhibitor among FDA-approved drugs that shows in vivo potential has significant implications for understanding autophagy modulation as a therapeutic strategy.

Text

2013-12-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/44591

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/