Oncogenic BRAF, unrestrained by TGFß-receptor signalling, drives right-sided colonic tumorigenesis.

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFß signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFß-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFß-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

Subversion of Niche-Signalling Pathways in Colorectal Cancer: What Makes and Breaks the Intestinal Stem Cell.

The intestinal epithelium fulfils pleiotropic functions in nutrient uptake, waste elimination, and immune surveillance while also forming a barrier against luminal toxins and gut-resident microbiota. Incessantly barraged by extraneous stresses, the intestine must continuously replenish its epithelial lining and regenerate the full gamut of specialized cell types that underpin its functions. Homeostatic remodelling is orchestrated by the intestinal stem cell (ISC) niche: a convergence of epithelial- and stromal-derived cues, which maintains ISCs in a multipotent state. Following demise of homeostatic ISCs post injury, plasticity is pervasive among multiple populations of reserve stem-like cells, lineage-committed progenitors, and/or fully differentiated cell types, all of which can contribute to regeneration and repair. Failure to restore the epithelial barrier risks seepage of toxic luminal contents, resulting in inflammation and likely predisposing to tumour formation. Here, we explore how homeostatic niche-signalling pathways are subverted in tumorigenesis, enabling ISCs to gain autonomy from niche restraints ("ISC emancipation") and transform into cancer stem cells capable of driving tumour initiation, progression, and therapy resistance. We further consider the implications of the pervasive plasticity of the intestinal epithelium for the trajectory of colorectal cancer, the emergence of distinct molecular subtypes, the propensity to metastasize, and the development of effective therapeutic strategies.

MCL1 Is Required for Maintenance of Intestinal Homeostasis and Prevention of Carcinogenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. METHODS: We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. RESULTS: Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. CONCLUSION: The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.

RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes.

Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.

Author Correction: Loss of BCL9/9l suppresses Wnt driven tumourigenesis in models that recapitulate human cancer.

The original version of this Article contained an error in the spelling of the author Miryam Müller, which was incorrectly given as Miryam Müeller. This has now been corrected in both the PDF and HTML versions of the Article.

Loss of BCL9/9l suppresses Wnt driven tumourigenesis in models that recapitulate human cancer.

Different thresholds of Wnt signalling are thought to drive stem cell maintenance, regeneration, differentiation and cancer. However, the principle that oncogenic Wnt signalling could be specifically targeted remains controversial. Here we examine the requirement of BCL9/9l, constituents of the Wnt-enhanceosome, for intestinal transformation following loss of the tumour suppressor APC. Although required for Lgr5+ intestinal stem cells and regeneration, Bcl9/9l deletion has no impact upon normal intestinal homeostasis. Loss of BCL9/9l suppressed many features of acute APC loss and subsequent Wnt pathway deregulation in vivo. This resulted in a level of Wnt pathway activation that favoured tumour initiation in the proximal small intestine (SI) and blocked tumour growth in the colon. Furthermore, Bcl9/9l deletion completely abrogated ß-catenin driven intestinal and hepatocellular transformation. We speculate these results support the just-right hypothesis of Wnt-driven tumour formation. Importantly, loss of BCL9/9l is particularly effective at blocking colonic tumourigenesis and mutations that most resemble those that occur in human cancer.

Intestinal Stem Cell Dynamics: A Story of Mice and Humans.

Stem cell dynamics define the probability of accumulating mutations within the intestinal epithelium. In this issue of Cell Stem Cell, Nicholson et al. (2018) report that human intestinal stem cell dynamics differ significantly from those of mice and establish that oncogenic mutations are more likely to expand; therefore, "normal" epithelium may carry multiple mutations.

TGFß pathway limits dedifferentiation following WNT and MAPK pathway activation to suppress intestinal tumourigenesis.

Recent studies have suggested increased plasticity of differentiated cells within the intestine to act both as intestinal stem cells (ISCs) and tumour-initiating cells. However, little is known of the processes that regulate this plasticity. Our previous work has shown that activating mutations of Kras or the NF-κB pathway can drive dedifferentiation of intestinal cells lacking Apc. To investigate this process further, we profiled both cells undergoing dedifferentiation in vitro and tumours generated from these cells in vivo by gene expression analysis. Remarkably, no clear differences were observed in the tumours; however, during dedifferentiation in vitro we found a marked upregulation of TGFß signalling, a pathway commonly mutated in colorectal cancer (CRC). Genetic inactivation of TGFß type 1 receptor (Tgfbr1/Alk5) enhanced the ability of KrasG12D/+ mutation to drive dedifferentiation and markedly accelerated tumourigenesis. Mechanistically this is associated with a marked activation of MAPK signalling. Tumourigenesis from differentiated compartments is potently inhibited by MEK inhibition. Taken together, we show that tumours arising in differentiated compartments will be exposed to different suppressive signals, for example, TGFß and blockade of these makes tumourigenesis more efficient from this compartment.

HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation.

Cancer genome sequencing projects have identified hundreds of genetic alterations, often at low frequencies, raising questions as to their functional relevance. One exemplar gene is HUWE1, which has been found to be mutated in numerous studies. However, due to the large size of this gene and a lack of functional analysis of identified mutations, their significance to carcinogenesis is unclear. To determine the importance of HUWE1, we chose to examine its function in colorectal cancer, where it is mutated in up to 15 per cent of tumours. Modelling of identified mutations showed that they inactivate the E3 ubiquitin ligase activity of HUWE1. Genetic deletion of Huwe1 rapidly accelerated tumourigenic in mice carrying loss of the intestinal tumour suppressor gene Apc, with a dramatic increase in tumour initiation. Mechanistically, this phenotype was driven by increased MYC and rapid DNA damage accumulation leading to loss of the second copy of Apc The increased levels of DNA damage sensitised Huwe1-deficient tumours to DNA-damaging agents and to deletion of the anti-apoptotic protein MCL1. Taken together, these data identify HUWE1 as a bona fide tumour suppressor gene in the intestinal epithelium and suggest a potential vulnerability of HUWE1-mutated tumours to DNA-damaging agents and inhibitors of anti-apoptotic proteins.