Sensitive High-Throughput Assays for Tumour Burden Reveal the Response of a Drosophila melanogaster Model of Colorectal Cancer to Standard Chemotherapies

Drosophila melanogaster (Drosophila) models of cancer are emerging as powerful tools to investigate the basic mechanisms underlying tumour progression and identify novel therapeutics. Rapid and inexpensive, it is possible to carry out genetic and drug screens at a far larger scale than in vertebrate organisms. Such whole-organism-based drug screens permits assessment of drug absorption and toxicity, reducing the possibility of false positives. Activating mutations in the Wnt and Ras signalling pathways are common in many epithelial cancers, and when driven in the adult Drosophila midgut, it induces aggressive intestinal tumour-like outgrowths that recapitulate many aspects of human colorectal cancer (CRC). Here we have taken a Drosophila CRC model in which tumourous cells are marked with both GFP and luciferase reporter genes, and developed novel high-throughput assays for quantifying tumour burden. Leveraging these assays, we find that the Drosophila CRC model responds rapidly to treatment with standard CRC-drugs, opening the door to future rapid genetic and drug screens.

Sensitive high-throughput assays for tumour burden reveal the response of a Drosophila melanogaster model of colorectal cancer to standard chemotherapies

Drosophila melanogaster (Drosophila) models of cancer are emerging as powerful tools to investigate the basic mechanisms underlying tumour progression and identify novel therapeutics. Rapid and inexpensive, it is possible to carry out genetic and drug screens at a far larger scale than in vertebrate organisms. Such whole-organism-based drug screens permits assessment of drug absorption and toxicity, reducing the possibility of false positives. Activating mutations in the Wnt and Ras signalling pathways are common in many epithelial cancers, and when driven in the adult Drosophila midgut, it induces aggressive intestinal tumour-like outgrowths that recapitulate many aspects of human colorectal cancer (CRC). Here we have taken a Drosophila CRC model in which tumourous cells are marked with both GFP and luciferase reporter genes, and developed novel high-throughput assays for quantifying tumour burden. Leveraging these assays, we find that the Drosophila CRC model responds rapidly to treatment with standard CRC-drugs, opening the door to future rapid genetic and drug screens.

A3 THE LOSS OF THE CIRCADIAN CLOCK GENE BMAL1 INCREASES TUMOUR INITIATION IN APCMIN MICE

Background Circadian rhythms are autonomously running 24h cycles in bodily processes. In animals these rhythms are driven by a molecular time keeper known as the circadian clock. The clock is a transcription-translation feedback loop composed of the transcription factors Bmal1 and Clock as well as their repressors Per and Cry. The circadian clock regulates over 40% of the genome rhythmically. Chronic circadian disruption, in the case of shift work, can lead to pathologies including cancer. Colorectal cancer is most frequently initiated through a mutation in the Wnt pathway regulator, Apc. Several studies have attempted to provide a mechanistic link between cancer and circadian clock disruption but the use of mice on mixed genetic backgrounds and poor circadian models have made this link unclear. Aims We aim to determine if the circadian clock plays a role in intestinal tumourigenesis. Methods We crossed the Apcmin mouse strain, a common intestinal tumour model, with Bmal1 mutant mice, which lack a functioning circadian clock. After creating an isogenic strain, we examined the number of tumours in control (Bmal1+/+) and clock dead (Bmal1-/-) animals. We derived organoids, a 3D cell culture method, from Apc+/+; Bmal1+/+ (healthy, clock-live), Apc+/+, Bmal1-/-(healthy, clock-dead), Apcmin; Bmal1+/+(adenoma, clock-live), Apcmin; Bmal1-/- (adenoma, clock-live) mouse ileum and collected every 2h from 24-48h after synchronizing their circadian clock. Collected samples were sent for RNA sequencing and assessed for circadian regulated transcripts. This experiment was followed up by in vitro organoid assays. Results The circadian clock controls 41 genes in the intestinal epithelium, including genes like Tead4 which are known to be important in intestinal biology. There are twofold more tumours in Bmal1-/- mice than their Bmal1+/+ littermates, and Bmal1-/- tumours upregulate Tead4 and Hippo pathway targets and downregulate Wnt pathway targets. Bmal1-/- adenoma organoids show increased self-renewal when compared to Bmal1+/+ adenoma organoids. However, this increase in self-renewal is lost when organoids are treated with inhibitors of the hippo pathway. Conclusions The circadian clock is important in maintaining the health of an organism, and disruption of the clock can lead to many health consequences including cancer. We show for the first time that the circadian clock controls the hippo signaling mediator Tead4. Additionally, we show that the loss of the clock leads to an increase in the number of tumours present in the epithelium which are characterized by an increase in hippo signaling. This research shows the important of considering time of day when studying stem cells during homeostasis and in cancer. Funding Agencies CIHRNSERC