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Mutations in TGFBR2, a component of the transforming growth factor (TGF)-β signaling pathway, occur in high-frequency microsatellite instability (MSI-H) colorectal cancer (CRC). In mouse models, Tgfbr2 inactivation in the intestinal epithelium accelerates the development of malignant intestinal tumors in combination with disruption of the Wnt-β-catenin pathway. However, no studies have further identified the genes influenced by TGFBR2 inactivation following disruption of the Wnt-β-catenin pathway. We previously described CDX2P-G19Cre;Apcflox/flox mice, which is stochastically null for Apc in the colon epithelium. In this study, we generated CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice, with simultaneous loss of Apc and Tgfbr2. These mice developed tumors, including adenocarcinoma in the proximal colon. We compared gene expression profiles between tumors of the two types of mice using microarray analysis. Our results showed that the expression of the murine homolog of GSDMC was significantly upregulated by 9.25-fold in tumors of CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice compared with those of CDX2P-G19Cre;Apcflox/flox mice. We then investigated the role of GSDMC in regulating CRC tumorigenesis. The silencing of GSDMC led to a significant reduction in the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GSDMC enhanced cell proliferation. These results suggested that GSDMC functioned as an oncogene, promoting cell proliferation in colorectal carcinogenesis. In conclusion, combined inactivation of both Apc and Tgfbr2 in the colon epithelium of a CRC mouse model promoted development of adenocarcinoma in the proximal colon. Moreover, GSDMC was upregulated by TGFBR2 mutation in CRC and promoted tumor cell proliferation in CRC carcinogenesis, suggesting that GSDMC may be a promising therapeutic target.

For over a century, fasting regimens have improved health, lifespan and tissue regeneration in diverse organisms, including humans 1 – 6 . However, how fasting and post-fast refeeding affect adult stem cells and tumour formation has yet to be explored in depth. Here we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation; post-fast refeeding augments the regenerative capacity of Lgr 5 + ISCs, and loss of the tumour suppressor gene Apc in post-fast-refed ISCs leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum-fed states, demonstrating that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust mTORC1 induction in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production or protein synthesis abrogates the regenerative or tumorigenic effects of post-fast refeeding. Given our findings, fast–refeeding cycles must be carefully considered and tested when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst in stem-cell-driven regeneration and tumorigenicity. Post-fast refeeding increases intestinal stem cell function and tumour formation by augmenting protein synthesis via polyamine metabolism.

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancer cases, leading to the synthesis of truncated APC products and the stabilization of β-catenin. Truncated APC is almost always retained in tumour cells, suggesting that it serves an essential function. Here, RNA interference has been used to down-regulate truncated APC in several colorectal cancer cell lines expressing truncated APCs of different lengths, thereby performing an analysis covering most of the mutation cluster region (MCR). The consequences on proliferation in vitro, tumour formation in vivo and the level and transcriptional activity of β-catenin have been investigated. Down-regulation of truncated APC results in an inhibition of tumour cell population expansion in vitro in 6 cell lines out of 6 and inhibition of tumour outgrowth in vivo as analysed in one of these cell lines, HT29. This provides a general rule explaining the retention of truncated APC in colorectal tumours and defines it as a suitable target for therapeutic intervention. Actually, we also show that it is possible to design a shRNA that targets a specific truncated isoform of APC without altering the expression of wild-type APC. Down-regulation of truncated APC is accompanied by an up-regulation of the transcriptional activity of β-catenin in 5 out of 6 cell lines. Surprisingly, the increased signalling is associated in most cases (4 out of 5) with an up-regulation of β-catenin levels, indicating that truncated APC can still modulate wnt signalling through controlling the level of β-catenin. This control can happen even when truncated APC lacks the β-catenin inhibiting domain (CiD) involved in targeting β-catenin for proteasomal degradation. Thus, truncated APC is an essential component of colorectal cancer cells, required for cell proliferation, possibly by adjusting β-catenin signalling to the \"just right\" level.

Loss of tumor suppressor adenomatous polyposis coli (APC) activates β-catenin to initiate colorectal tumorigenesis. However, β-catenin (CTNNB1) activating mutations rarely occur in human colorectal cancer (CRC). We found that APC loss also results in up-regulation of IL-6 signal transducer (IL-6ST/gp130), thereby activating Src family kinases (SFKs), YAP, and STAT3, which are simultaneously up-regulated in the majority of human CRC. Although, initial YAP activation, which stimulates IL6ST gene transcription, may be caused by reduced serine phosphorylation, sustained YAP activation depends on tyrosine phosphorylation by SFKs, whose inhibition, along with STAT3-activating JAK kinases, causes regression of established colorectal tumors. These results explain why APC loss is a more potent initiating event than the mere activation of CTNNB1.

Many epithelial stem cell populations follow a pattern of stochastic stem cell divisions called 'neutral drift'. It is hypothesised that neutral competition between stem cells protects against the acquisition of deleterious mutations. Here we use a Porcupine inhibitor to reduce Wnt secretion at a dose where intestinal homoeostasis is maintained despite a reduction of Lgr5+ stem cells. Functionally, there is a marked acceleration in monoclonal conversion, so that crypts become rapidly derived from a single stem cell. Stem cells located further from the base are lost and the pool of competing stem cells is reduced. We tested whether this loss of stem cell competition would modify tumorigenesis. Reduction of Wnt ligand secretion accelerates fixation of Apc -deficient cells within the crypt leading to accelerated tumorigenesis. Therefore, ligand-based Wnt signalling influences the number of stem cells, fixation speed of Apc mutations and the speed and likelihood of adenoma formation. Wnt ligands are essential for intestinal homoeostasis and stem cell maintenance. Here, the authors show that reduction in Wnt secretion reduces the number of intestinal stem cells; this results in rapid fixation of mutated stem cells and accelerated adenoma formation due to lack of cell competition.

Nonstop extension mutations, a.k.a. stop-lost or stop-loss mutations, convert a stop codon into a sense codon resulting in translation into the 3’ untranslated region until the next in-frame stop codon, thereby extending the C-terminus of a protein. In cancer, only nonstop mutations in SMAD4 have been functionally characterized, while the impact of other nonstop mutations remain unknown. Here, we exploit our pan-cancer NonStopDB dataset and test all 2335 C-terminal extensions arising from somatic nonstop mutations in cancer for their impact on protein expression. In a high-throughput screen, 56.1% of the extensions effectively reduce protein abundance. Extensions of multiple tumor suppressor genes like PTEN , APC , B2M , CASP8 , CDKN1B and MLH1 are effective and validated for their suppressive impact. Importantly, the effective extensions possess a higher hydrophobicity than the neutral extensions linking C-terminal hydrophobicity with protein destabilization. Analyzing the proteomes of eleven different species reveals conserved patterns of amino acid distribution in the C-terminal regions of all proteins compared to the proteomes like an enrichment of lysine and arginine and a depletion of glycine, leucine, valine and isoleucine across species and kingdoms. These evolutionary selection patterns are disrupted in the cancer-derived effective nonstop extensions. Nonstop extension mutations in cancer remain poorly characterized. Here, the authors test the impact of 2335 tumor-derived C-terminal nonstop extensions on protein expression and find the majority to be suppressive; they also analyze their biochemical properties and relation to evolutionary selection patterns.

Wnt signalling is prevented by the proteosomal degradation of β-catenin, which occurs in a destruction complex containing adenomatous polyposis coli (APC), APC-like (APCL), Axin and Axin2. Truncating mutations of the APC gene result in the constitutive stabilisation of β-catenin and the initiation of colon cancer, although tumour cells tolerate the expression of wild-type APCL. Using the colocalisation of overexpressed Axin, APC and APCL constructs as a readout of interaction, we found that Axin interacted with the second twenty amino acid repeat (20R2) of APC and APCL. This interaction involved a domain adjacent to the C-terminal DIX domain of Axin. We identified serine residues within the 20R2 of APCL that were involved in Axin colocalisation, the phosphorylation of truncated APCL and the down-regulation of β-catenin. Our results indicated that Axin, but not Axin2, displaced APC, but not APCL, from the cytoskeleton and stimulated its incorporation into bright cytoplasmic dots that others have recognised as β-catenin destruction complexes. The SAMP repeats in APC interact with the N-terminal RGS domain of Axin. Our data showed that a short domain containing the first SAMP repeat in truncated APC was required to stimulate Axin oligomerisation. This was independent of Axin colocalisation with 20R2. Our data also suggested that the RGS domain exerted an internal inhibitory constraint on Axin oligomerisation. Considering our data and those from others, we discuss a working model whereby β-catenin phosphorylation involves Axin and the 20R2 of APC or APCL and further processing of phospho-β-catenin occurs upon the oligomerisation of Axin that is induced by binding the SAMP repeats in APC.

Canonical Wnt signaling is critical for the control of osteoblast differentiation in human mesenchymal stem cells. MicroRNAs (miRs) are essential regulators of cell differentiation by post-transcriptional regulation of target gene expression. The aim of the present study was to investigate the molecular mechanism by which miR-142-3p promotes osteoblastic differentiation using the human fetal osteoblastic 1.19 (hFOB1.19), real-time PCR and western blot analysis. Results showed an increased expression of miR-142-3p during osteoblast differentiation in the mesenchymal precursor cell line, hFOB1.19. In addition, the ectopic overexpression of miR-142-3p promoted hFOB1.19 differentiation, whereas the inhibition of miR-142-3p repressed differentiation. The expression of miR-142-3p was positively correlated with β-catenin, an important protein in Wnt signaling. The adenomatous polyposis coli (APC) gene was a direct target of miR-142-3p, whereby miR-142-3p promoted Wnt signaling through inhibition of APC, leading to accumulation and nuclear translocation of β-catenin. Therefore, miR-142-3p may be an essential mediator of osteoblast differentiation and a new therapeutic strategy for osteogenesis disorders.

MicroRNAs (miRNAs) are important regulators of stem and progenitor cell functions. We previously reported that miR-142 and miR-150 are upregulated in human breast cancer stem cells (BCSCs) as compared to the non-tumorigenic breast cancer cells. In this study, we report that miR-142 efficiently recruits the APC mRNA to an RNA-induced silencing complex, activates the canonical WNT signaling pathway in an APC-suppression dependent manner, and activates the expression of miR-150. Enforced expression of miR-142 or miR-150 in normal mouse mammary stem cells resulted in the regeneration of hyperproliferative mammary glands in vivo. Knockdown of endogenous miR-142 effectively suppressed organoid formation by BCSCs and slowed tumor growth initiated by human BCSCs in vivo. These results suggest that in some tumors, miR-142 regulates the properties of BCSCs at least in part by activating the WNT signaling pathway and miR-150 expression. Messenger RNA molecules take the information encoded in a gene's DNA sequence and turn it into instructions for building a protein. However, if certain smaller molecules of RNA—called microRNAs—bind to a messenger RNA molecule, they ‘silence’ it, which prevents the information in the messenger RNA from being translated to make a protein. Despite their small size, microRNAs are very powerful. These molecules are able to simultaneously inhibit the translation of hundreds of messenger RNAs and perform many roles, including controlling cell growth and maintaining populations of stem cells. Furthermore, microRNAs have been linked to different aspects of the growth of cancerous cells. Certain microRNAs appear to suppress tumors by regulating the growth of the stem cells found there, while others appear to be ‘hyperactive’ in cancers—including breast cancer, colon cancer, and blood cancer. In 2009, researchers compared the amount of microRNA in breast cancer stem cells that are highly capable of forming tumors with the amount in other cancer cells within the same tumor. Amongst other differences, two microRNAs (called miR-142 and miR-150) were found to be hyperactive in human breast cancer stem cells. One of them, miR-142, is known to target a gene called APC that inhibits the renewal of normal stem cells. Mutations in the APC gene have been linked to colon cancer, and scientists have suggested that the mutations inactivate APC in cancer cells to promote unregulated cell growth. Breast tumors rarely have mutations in the APC gene, but Isobe et al. wondered whether microRNAs that target this gene might also promote the growth of these tumor cells. Isobe et al.—including several of the researchers involved in the 2009 work—show that miR-142 does target the APC gene in human breast cancer stem cells, and silences it. With the gene silenced, a cancer-promoting pathway turns on and more miR-150 is made. Increasing the amount of either miR-142 or miR-150 causes excessive cell growth in breast tissue and can form abnormal breast tissue in mice. Reducing the amount of miR-142 in human breast cancer stem cells slows the growth of breast tumors. Although they only make up a small population of human breast cancer cells, focusing on breast cancer stem cells could uncover the cancer-promoting pathways that are activated in human breast cancers.

Summary Mutations in the adenomatous polyposis coli ( APC ) tumor suppressor gene strongly predispose to development of gastro-intestinal tumors. Central to the tumorigenic events in APC mutant cells is the uncontrolled stabilization and transcriptional activation of the protein β-catenin. Many questions remain as to how APC controls β-catenin degradation. Remarkably, the large C-terminal region of APC, which spans over 2000 amino acids and includes critical regions in downregulating β-catenin, is predicted to be natively unfolded. Here we discuss how this uncommonly large disordered region may help to coordinate the multiple cellular functions of APC. Recently, a significant number of germline and somatic missense mutations in the central region of APC were linked to tumorigenesis in the colon as well as extra-intestinal tissues. We classify and localize all currently known missense mutations in the APC structure. The molecular basis by which these mutations interfere with the function of APC remains unresolved. We propose several mechanisms by which cancer-related missense mutations in the large disordered domain of APC may interfere with tumor suppressor activity. Insight in the underlying molecular events will be invaluable in the development of novel strategies to counter dysregulated Wnt signaling by APC mutations in cancer.