Background & Aims Aspirin reduces the incidence of and mortality from colorectal cancer (CRC) by unknown mechanisms. Cancer cells have defects in signaling via the mechanistic target of rapamycin (mTOR), which regulates proliferation. We investigated whether aspirin affects adenosine monophosphate–activated protein kinase (AMPK) and mTOR signaling in CRC cells. Methods The effects of aspirin on mTOR signaling, the ribosomal protein S6, S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) were examined in CRC cells by immunoblotting. Phosphorylation of AMPK was measured; the effects of loss of AMPKα on the aspirin-induced effects of mTOR were determined using small interfering RNA (siRNA) in CRC cells and in AMPK^{α1/α2−/−} mouse embryonic fibroblasts. LC3 and ULK1 were used as markers of autophagy. We analyzed rectal mucosa samples from patients given 600 mg aspirin, once daily for 1 week. Results Aspirin reduced mTOR signaling in CRC cells by inhibiting the mTOR effectors S6K1 and 4E-BP1. Aspirin changed nucleotide ratios and activated AMPK in CRC cells. mTOR was still inhibited by aspirin in CRC cells after siRNA knockdown of AMPKα, indicating AMPK-dependent and AMPK-independent mechanisms of aspirin-induced inhibition of mTOR. Aspirin induced autophagy, a feature of mTOR inhibition. Aspirin and metformin (an activator of AMPK) increased inhibition of mTOR and Akt, as well as autophagy in CRC cells. Rectal mucosal samples from patients given aspirin had reduced phosphorylation of S6K1 and S6. Conclusions Aspirin is an inhibitor of mTOR and an activator of AMPK, targeting regulators of intracellular energy homeostasis and metabolism. These could contribute to its protective effects against development of CRC.

Substantial evidence indicates nonsteroidal anti-inflammatory drugs (NSAIDs) protect against colorectal cancer (CRC). However, the molecular basis for this anti-tumour activity has not been fully elucidated. We previously reported that aspirin induces signal-specific IκBα degradation followed by NFκB nuclear translocation in CRC cells, and that this mechanism contributes substantially to aspirin-induced apoptosis. We have also reported the relative specificity of this aspirin-induced NFκB-dependent apoptotic effect for CRC cells, in comparison to other cancer cell types. It is now important to establish whether there is heterogeneity within CRC, with respect to the effects of aspirin on the NFκB pathway and apoptosis. p53 signalling and DNA mismatch repair (MMR) are known to be deranged in CRC and have been reported as potential molecular targets for the anti-tumour activity of NSAIDs. Furthermore, both p53 and MMR dysfunction have been shown to confer resistance to chemotherapeutic agents. Here, we set out to determine the p53 and hMLH1 dependency of the effects of aspirin on NFκB signalling and apoptosis in CRC. We specifically compared the effects of aspirin treatment on cell viability, apoptosis and NFκB signalling in an HCT-116 CRC cell line with the p53 gene homozygously disrupted (HCT-116^p53−/−) and an HCT-116 cell line rendered MMR proficient by chromosomal transfer (HCT-116^+ch3), to the parental HCT-116 CRC cell line. We found that aspirin treatment induced apoptosis following IκBα degradation, NFκB nuclear translocation and repression of NFκB-driven transcription, irrespective of p53 and DNA MMR status. These findings are relevant for design of both novel chemopreventative agents and chemoprevention trials in CRC.

Cancer cell plasticity enables the acquisition of new phenotypic features and is implicated as a major driver of metastatic progression^1,2. Metastasis occurs mostly in the absence of additional genetic alterations^3–5, which suggests that epigenetic mechanisms are important⁶. However, they remain poorly defined. Here we identify the chromatin-remodelling enzyme ATRX as a key regulator of colonic lineage fidelity and metastasis in colorectal cancer. Atrx loss promotes tumour invasion and metastasis, concomitant with a loss of colonic epithelial identity and the emergence of highly plastic mesenchymal and squamous-like cell states. Combined analysis of chromatin accessibility and enhancer mapping identified impairment of activity of the colonic lineage-specifying transcription factor HNF4A as a key mediator of these observed phenotypes. We identify squamous-like cells in human patient samples and a squamous-like expression signature that correlates with aggressive disease and poor patient prognosis. Collectively, our study defines the epigenetic maintenance of colonic epithelial identity by ATRX and HNF4A as suppressors of lineage plasticity and metastasis in colorectal cancer.

Faecal immunochemical test (FIT)-directed pathways based on a single test have been implemented for symptomatic patients. However, with a single test, the sensitivity is 87 per cent at 10 µg haemoglobin (Hb) per g faeces. This aims of this study were to define the diagnostic performance of a single FIT, compared with double FIT in symptomatic populations. Two sequential prospective patient cohorts referred with symptoms from primary care were studied. Patients in cohort 1 were sent a single FIT, and those in cohort 2 received two tests in succession before investigation. All patients were investigated, regardless of having a positive or negative test (threshold 10 µg Hb per g). In cohort 1, 2260 patients completed one FIT and investigation. The sensitivity of single FIT was 84.1 (95 per cent c.i. 73.3 to 91.8) per cent for colorectal cancer and 67.4 (61.0 to 73.4) per cent for significant bowel pathology. In cohort 2, 3426 patients completed at least one FIT, and 2637 completed both FITs and investigation. The sensitivity of double FIT was 96.6 (90.4 to 99.3) per cent for colorectal cancer and 83.0 (77.4 to 87.8) per cent for significant bowel pathology. The second FIT resulted in a 50.0 per cent reduction in cancers missed by the first FIT, and 30.0 per cent for significant bowel pathology. Correlation between faecal Hb level was only modest (r_s = 0.58), and 16.8 per cent of double tests were discordant, 11.4 per cent in patients with colorectal cancer and 18.3 per cent in those with significant bowel pathology. FIT in patients with high-risk symptoms twice in succession reduces missed significant colorectal pathology and has an acceptable workload impact.

Aspirin reduces colorectal cancer (CRC) incidence and mortality. Understanding the biology responsible for this protective effect is key to developing biomarker-led approaches for rational clinical use. Wnt signaling drives CRC development from initiation to progression through regulation of epithelial-mesenchymal transition (EMT) and cancer stem cell populations. Here, we investigated whether aspirin can rescue these proinvasive phenotypes associated with CRC progression in Wnt-driven human and mouse intestinal organoids. We evaluated aspirin-mediated effects on phenotype and stem cell markers in intestinal organoids derived from mouse (ApcMin/+ and Apcflox/flox) and human familial adenomatous polyposis patients. CRC cell lines (HCT116 and Colo205) were used to study effects on motility, invasion, Wnt signaling, and EMT. Aspirin rescues the Wnt-driven cystic organoid phenotype by promoting budding in mouse and human Apc deficient organoids, which is paralleled by decreased stem cell marker expression. Aspirin-mediated Wnt inhibition in ApcMin/+ mice is associated with EMT inhibition and decreased cell migration, invasion, and motility in CRC cell lines. Chemical Wnt activation induces EMT and stem-like alterations in CRC cells, which are rescued by aspirin. Aspirin increases expression of the Wnt antagonist Dickkopf-1 in CRC cells and organoids derived from familial adenomatous polyposis patients, which contributes to EMT and cancer stem cell inhibition. We provide evidence of phenotypic biomarkers of response to aspirin with an increased epithelial and reduced stem-like state mediated by an increase in Dickkopf-1. This highlights a novel mechanism of aspirin-mediated Wnt inhibition and potential phenotypic and molecular biomarkers for trials.