Colorectal cancer is one of the most common forms of cancer in the world, with more than half a million new cases annually. Amongst the most promising new therapies, niclosamide—an FDA-approved drug for treating tapeworm infections—is being assessed in a stage II clinical trial for the treatment of metastatic colorectal cancer. Despite this advanced stage of research, the underlying mechanisms behind its actions remain uncertain. Niclosamide reduces the growth of colorectal cancer cells by targeting several intracellular signalling pathways, including the β-catenin-dependent WNT signalling pathway. In a recent paper published in the Biochemical Journal [Biochem. J. (2019) 476, 535–546], Wang and colleagues revealed that niclosamide down-regulates β-catenin-dependent WNT signalling in colorectal cancer cells by degrading components of the pathway via autophagy. Autophagy is a catabolic process in which cellular macromolecules and organelles are recycled to their monomer units. This finding provides a further understanding of the actions of niclosamide upon colorectal cancer cells and may yield improved future treatment models for colorectal cancer patients.
Colorectal cancer (also known as bowel cancer) is the third most prevalent cancer in the world and, according to the most recent Cancer Research UK statistics, accounts for more than 16 000 deaths and more than 40 000 new cases in the U.K. every year . The underlying causes of colorectal cancer are complex and multi-staged; risk factors include aging, sex (increased risk with being male), and lifestyle, with underlying genetics significant to a lesser extent .
Colorectal cancer affects the digestive tract from the small intestine to the rectum. It can begin with polyps in the digestive tract, which can gradually become larger, attract blood vessels, and become metastatic, allowing it to spread to other tissues . Treatments depend on the severity of cancer and its developmental stage, ranging from surgery, radiotherapy, chemotherapy, and the use of antibodies.
At the cellular level, a key pathway involved in the growth of colorectal cancer cells is β-catenin-dependent WNT signalling (previously referred to as ‘canonical WNT signalling’, and hereafter ‘WNT/β-catenin signalling’). Mutations in WNT/β-catenin signalling are thought to be a major driving force in colorectal cancer development, which is hyper-activated in 80% of cases . Thus, pharmaceuticals that can inhibit this pathway are highly desirable.
In 2009, a group from Duke University Medical Centre discovered that the widely used anthelmintic drug, niclosamide, down-regulated the activity of the WNT/β-catenin pathway in vitro . They found that niclosamide stimulated the internalization of Frizzled receptor 1 (Fzd1), a WNT-ligand receptor, into unidentified puncta . Since then, several studies by the same authors and by others have reported that niclosamide (and its derivatives) inhibits colorectal cancer development both in vitro and in vivo . This includes those with mutations in the key down-stream WNT/β-catenin signalling inhibitor, adenomatous polyposis coli (APC), an integral part of the β-catenin-destruction complex . In their most recent work, published in , the authors sought to reveal the underlying mechanisms of niclosamide action and found that components of the WNT/β-catenin pathway are targeted for degradation within the cell through the mechanism of macroautophagy (hereafter referred to as autophagy).
Autophagy is an intracellular process that degrades macromolecules and organelles into basic building blocks (such as amino acids), which can then be recycled into new macromolecules. It is a system that runs at basal level in all cells but the rate of autophagy increases during times of inflammation, hypoxia, stress, or low nutrition to provide an additional energy source for the cell . Upon autophagy induction, double-membraned vesicles, called autophagosomes, engulf large parts of the cytoplasm. Autophagosomes fuse with acidic, enzyme-rich lysosomes to degrade their contents. The mammalian target of rapamycin complex 1 (mTORC1) is the major regulator of autophagy. Activated mTORC1 inhibits autophagy by disrupting autophagosome formation .
Paradoxically, although autophagy is beneficial to maintain homeostasis in healthy cells, its over-activity has been implicated in a distinctive form of cell death, called autophagic cell death . Autophagic cell death is typically identified histologically, by the presence of large autophagosomes within the dying cell. Autophagic cell death remains a controversial topic since these autophagosomes may represent ongoing autophagy coinciding with cell death. However, specific ablation of autophagy-related genes was recently shown to reduce cell death, suggesting that autophagy itself can induce cell death, at least in some situations. Therefore, activating autophagy using small molecules could be an ideal way to stimulate continuous autophagic activity and thereby induce autophagic cell death. However, it should be reiterated that a complete understanding of this aspect of autophagy is lacking .
Wang and colleagues  have now revealed that the internalized Fzd1 puncta that appear upon niclosamide treatment are positive for a marker of the autophagosome called LC3. The researchers showed that niclosamide acts via autophagy in colorectal cancer cells by blocking its actions using pharmacological inhibitors of autophagy and genetic manipulation. These are exciting data regarding the mechanism of how niclosamide reduces WNT/β-catenin signalling in colorectal cancer and provides a further understanding of WNT/β-catenin signalling inactivation. The findings are in agreement with the ability of niclosamide to stimulate autophagy in other types of the cell [9,10]. However, since niclosamide can cause ubiquitination of many proteins in other cancer cell types , it is unlikely that Fzd1 and β-catenin are specifically targeted for autophagic degradation.
The aforementioned study by Wang and colleagues leads to some intriguing questions. For example, as well as WNT/β-catenin signalling, niclosamide also affected the autophagy and mTORC1 pathways. In line with this observation, it might appear that a more precise approach would be to target WNT/β-catenin signalling specifically, to avoid affecting mTORC1 and autophagy. However, could the inhibition of WNT/β-catenin signalling by niclosamide be secondary to its actions on mTORC1 and/or autophagy? mTORC1 is a hub for anabolic and catabolic signals, and its activation can be involved in many types of cancer [7,11]. Indeed, a study in which colorectal cancer cells were treated with the mTOR inhibitor AZD-2014 (which inhibits both mTORC1 and mTORC2 function) was so effective at blocking mTORC1 activity, that it led to continuous autophagy and eventually autophagic cell death . This treatment was effective both in vitro and in vivo, improving the survival rate of mice with human colon cancer xenografts . Furthermore, mTORC1 ablation (by excising an integral region of the mTORC1 scaffolding protein, Raptor) could prevent colorectal cancer development caused by hyper-activation of the WNT/β-catenin signalling pathway following genetic ablation of APC, in vivo . Since niclosamide is a strong mTORC1 inhibitor in colorectal cancer, may ubiquitinate many targets , and can block multiple signalling pathways , it is important to understand if the main benefits of niclosamide come via modulating mTORC1 signalling pathway.
One further consideration is that niclosamide has been shown to control mTORC1 activity by altering intracellular pH, possibly by stimulating proton release from lysosomes . However, maintaining an acidic pH in lysosomes is critical for the activity of the lysosomal enzymes, thereby under some circumstances, niclosamide can inhibit autophagy . Wang and colleagues found that the actions of niclosamide in colorectal cancer cells were blocked by inhibiting autophagy using pharmacological inhibitors or gene ablation, suggesting that niclosamide alone did not abolish autophagy in their experiments. However, if niclosamide does affect lysosomal pH then different niclosamide concentrations may entirely block degradation by autophagy. These dose-dependent dual abilities of niclosamide have been observed in several other cell types .
Interestingly, Wang and colleagues found that not all of the colorectal cancer cell lines responded to niclosamide. Since there is a broad spectrum of sub-types that comprise colorectal cancer , it is critical to unpick the reasons why niclosamide was ineffective in certain cell lines but not in others; such findings may in the future be applied to treatment of human cancers, allowing one to predict if niclosamide would be beneficial to particular patients.
In summary, recent research by Wang and colleagues has shown that niclosamide inhibits WNT/β-catenin signalling in colorectal cancer cells by degrading components of the pathway via autophagy. It should be made clear that the relevance of the current data to patients is tempered by the fact that these experiments were performed in cell lines. Nevertheless, since niclosamide is currently being tested in phase II clinical trial, further understanding of how niclosamide functions at the basic research level is extremely timely.
The author is very grateful to Dr Thibault Bouderlique (Department of Laboratory Medicine, Karolinska Institute, Sweden) for critical reading, and to Prof. Lars Sävendahl (Department of Women's and Children's Health, Karolinska Institutet and Pediatric Endocrinology Unit, Karolinska University Hospital) for financial support. P.T.N. was financially supported by Stiftelsen Frimurare Barnhuset and Sällskapet Barnavård.
The Author declares that there are no competing interests associated with this manuscript.