Pleiotropic role of NOTUM in colorectal cancer

Colorectal cancer (CRC) is one of the most common malignancies worldwide and a leading cause of cancer-related deaths. The vast majority of CRCs harbour a mutation in tumour suppressor gene adenomatous polyposis coli (APC), resulting in the continuous activation of the Wnt pathway.¹ The subsequent translocation of β-catenin to the nucleus induces Wnt target gene expression that is regulating many key cellular processes including differentiation, stem cell self-renewal and proliferation. Given the importance of the Wnt pathway in homeostatic regulation, this pathway is tightly controlled to avoid excess signalling and thereby promoting tissue overgrowth or tumour initiation. Therefore, Wnt activity induces a negative feedback loop that facilitates the expression of negative Wnt regulators, including the Notum gene encoding the extracellular palmitoleoyl-protein carboxylesterase protein.²

Notum was first discovered in the Drosophila model organism, where a screen for genes interacting with wingless (wg) signalling, a pathway conserved in mammals known as the Wnt pathway, demonstrated that overexpression of Notum resulted in reduced wing sizes indicating a wg inhibitory role.³ Extensive follow-up research on Notum’s inhibitory mechanism revealed that Notum directly exerts its effect by removing the palmitoleate moiety on Wnt ligands, which is essential for signalling as it contributes to the interaction with Frizzled receptors.² The deacylating capacity is dependent on glypicans, heparin sulphate proteoglycans that are bound to Wnts, which co-localise Notum and Wnts at the cell surface.² Also in mammals, Notum has become a major topic of interest in recent years. In particular, the role of NOTUM within the intestinal stem cell (ISC) niche is extensively studied. For instance, a previous study demonstrated that NOTUM negatively regulates the number of ISCs in the homeostatic gut. More specifically, on ageing, Paneth cells populating the ISC niche increase their production of NOTUM by a process involving mTORC1 and PPAR-alpha signalling, thereby countering the stem cell promoting function of Wnt ligands.⁴ This subsequently results in a reduction of functional ISC numbers that compete for niche occupation, which can have critical consequences for intestinal tumour initiation.

As the intestinal epithelium has an extremely high turnover rate, the ISCs are specifically prone to mutational insults. When an ISC acquires an oncogenic mutation that confers a competitive advantage to the cell, its offspring can replace all neighbouring wild-type ISCs and the mutant clone can take over the entire crypt.⁵ As this is a probabilistic process, the rate of crypt take over is dependent on the number of competing ISCs,⁶ with less functional ISCs resulting in an increased rate of monoclonal conversion. Given the crucial role of NOTUM in controlling ISC numbers in the ageing intestine, it comes as no surprise that NOTUM also plays an active role during intestinal tumour formation. Indeed, recent work in mice demonstrated that Apc-mutant ISCs actively disadvantage wild-type ISCs by the secretion of a range of Wnt antagonists, including NOTUM.^{7 8} The presence of oncogenic NOTUM in the crypt bottom confers a ‘supercompetitor’ phenotype to the Apc-mutant cells by driving differentiation of wild-type ISCs thereby facilitating the outgrowth of mutant clones and the development of premalignant adenomas.^{7 8} Moreover, pharmacological inhibition of NOTUM or simultaneous deletion of Apc and Notum, both result in reduced mutant crypt fixation and decreased tumour burden and hence NOTUM was identified as a key regulator of intestinal tumour initiation.^{7 9}

The role of NOTUM in tumourigenesis is more complex than initially thought. Although primarily assumed that Apc-specific upregulation of NOTUM is solely a consequence of a negative feedback response to hyperactivated Wnt signalling, a recent study by Tian et al now reveals a novel role for Notum, and that it can serve as both a tumour suppressor and oncogene depending on tumour stage.¹⁰ More specifically, they demonstrate that NOTUM inhibition in vivo promotes outgrowth of established adenomas while decreasing metastatic potential of advanced colorectal tumours, indicating a more versatile role for NOTUM in CRC. The opposing effects of NOTUM inhibition at the various stages of CRC progression can be attributed to a genetic switch that is caused by concomitant loss of Apc and Tp53 function.¹⁰ The dual function of NOTUM can be accounted for by differential glypican activity. As glypicans act on the cell surface level, it is hypothesised that on cleavage by NOTUM, glypicans either become neutralised or start to negatively regulate receptor activity through ligand competition. As such, in Apc-mutant tumouroids, organoids harvested from tumour tissue, NOTUM exerts its tumour suppressive effects by cleaving glypican-1, thereby inhibiting mTORC1 signalling. On additional loss of Tp53, NOTUM becomes oncogenic by cleaving glypican-4 that normally exerts tumour suppressive effects by negatively regulating pro-tumourigenic TGFβ activity.¹⁰

Inhibition of NOTUM has marked consequences for intestinal tumourigenesis. The most striking effects were observed using NOTUM inhibition in orthotopically implanted mouse tumouroids of advanced CRC, which resulted in growth arrest, decreased metastasis, and a 100% survival advantage in mice.¹⁰ These new insights reveal an interesting therapeutic potential for NOTUM inhibition in patients with advanced stage CRC, particularly when aiming to prevent metastasis. Importantly, when implementing novel anticancer therapies based on NOTUM inhibition in the future, one should stratify patients based on the TP53 status, as the oncogenic phenotype of NOTUM is a direct consequence of TP53 mutations.¹⁰ Critically, the tumour suppressive role of Notum at the adenoma stage remains ambiguous, which warrents the use of NOTUM inhibition in early stage CRC, as well as in patients with advanced CRC harbouring synchronous cancer and polyps. More specifically, the complex interplay between autonomous and non-autonomous effects of NOTUM appears hard to disentangle, as NOTUM inhibition has been demonstrated to both decrease and promote intestinal tumour formation in distinct Apc-mutant models, either by inhibiting supercompetition^{7 9} or endogenously promoting Apc-mutant cells,¹⁰ respectively. In addition, this complexity increases even further as established adenomas display heterogeneous expression of Notum, suggestive of clonal evolution, and long-term NOTUM inhibition promotes the expression of even more negative regulators of the Wnt pathway.⁹

Together, these studies highlight the pleiotropic role of NOTUM in cancer initiation, progression and metastasis, and emphasise the need to further define its role in directly modulating Wnt signalling and regulating other critical signalling pathways via a diverse set of glypicans.

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