Nutrient-dependent effects of the autophagy-lysosomal inhibitor, Chloroquine, on cell death and lysosomal functionality

There is huge potential for targeting pro-survival autophagy as a cancer therapeutic strategy, but this approach has not yet been fully realised due to the complex involvement of autophagy for cancer biology. In early stages of transformation, autophagy acts as a tumour suppressor, whilst in a developed tumour, autophagy aids cancer cell survival contributing to resistance. This thesis aimed to interrogate the role of autophagy for survival in metastatic breast cancer using the 4T1 mouse mammary carcinoma model. Investigations utilised the autophagy-lysosomal inhibitor Chloroquine (CQ) since this compound is currently in clinical trials for a variety of blood and solid cancers, including breast cancer. Here, the mechanisms of CQ and furthermore, the potential of combining this drug with metabolic targeting strategies were investigated. In doing this, an unexpected resistance mechanism linking glucose metabolism and CQ was uncovered. In clonogenic assays, CQ induced cell death that cooperated with other therapeutic stressors such as ionising irradiation and PI3K-Akt inhibition. CQ and the metabolic stress of serum starvation produced cell death within 24hrs; however, unexpectedly, further glucose starvation or hexokinase inhibition fully rescued cell viability. The cytotoxic effects of CQ were found to be autophagy-independent as knockdown of ATG proteins did not mimic CQ. As the form of cell death in our model did not resemble classical caspase-dependent apoptosis or necrosis, it was hypothesised the cytotoxic effects of CQ were potentially triggering lysosome membrane permeabilisation. Indeed, CQ treatment did lead to marked lysosomal stress and enlargement, suggestive of LMP. In contrast, while CQ was still able to enter and deacidify the lysosome in glucose starved cells, it failed to induce enlargement. Our data indicate that glucose metabolic rate has a profound influence on the efficacy of CQ to target lysosomes and to induce LMP-mediated death. These effects may be reducing clinical outcomes of CQ in cancer cells with reduced glucose metabolism.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral-pg

Grantor dc:publisher.institution

University of Strathclyde

Year dc:date.issued

2017

Author dc:creator

• Gallagher, Laura

DOI

10.48730/4txq-ma06

Identifier

T14524

Author Identifier

201252093

OAI identifier oai:identifier

oai:strathclyde:zw12z537n