[Show abstract][Hide abstract] ABSTRACT: Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1-S6K-eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.
[Show abstract][Hide abstract] ABSTRACT: The loss of Apc, causing Wnt-mediated epithelial proliferation, is an early event in colorectal cancer (CRC) development. This hyperproliferative state requires signalling though the mTOR pathway, with the current paradigm suggesting that upregulation of translation initiation via phosphorylation of 4EBP1 is crucial. This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 function, would be ineffective in limiting development and progression of intestinal adenomas.
Gut 06/2014; 63(Suppl 1):A130-A131. · 10.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The non-receptor tyrosine kinase c-Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20% of the human tumours, although this has never been addressed due to multiple redundant family members. Here, we have used the adult Drosophila and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage-induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, we demonstrate that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self-renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non-redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, we show that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, we demonstrate a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation in vivo.
[Show abstract][Hide abstract] ABSTRACT: MicroRNA deregulation is frequent in human colorectal cancers (CRCs), but little is known as to whether it represents a bystander event or actually drives tumor progression in vivo. We show that miR-135b overexpression is triggered in mice and humans by APC loss, PTEN/PI3K pathway deregulation, and SRC overexpression and promotes tumor transformation and progression. We show that miR-135b upregulation is common in sporadic and inflammatory bowel disease-associated human CRCs and correlates with tumor stage and poor clinical outcome. Inhibition of miR-135b in CRC mouse models reduces tumor growth by controlling genes involved in proliferation, invasion, and apoptosis. We identify miR-135b as a key downsteam effector of oncogenic pathways and a potential target for CRC treatment.
Cancer cell 04/2014; 25(4):469-83. · 25.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Self-renewal is essential for multicellular organisms but carries the risk of somatic mutations that can lead to cancer, which is particularly critical for rapidly renewing tissues in a highly mutagenic environment such as the intestinal epithelium. Using computational modeling and in vivo experimentation, we have analyzed how adenomatous polyposis coli (APC) mutations and β-catenin aberrations affect the maintenance of mutant cells in colonic crypts. The increasing abundance of APC along the crypt axis forms a gradient of cellular adhesion that causes more proliferative cells to accelerate their movement toward the top of the crypt, where they are shed into the lumen. Thus, the normal crypt can efficiently eliminate β-catenin mutant cells, whereas APC mutations favor retention. Together, the molecular design of the APC/β-catenin signaling network integrates cell proliferation and migration dynamics to translate intracellular signal processing and protein gradients along the crypt into intercellular interactions and whole-crypt physiological or pathological behavior.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.Cell Death and Differentiation advance online publication, 28 February 2014; doi:10.1038/cdd.2014.15.
Cell death and differentiation 02/2014; · 8.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although somatic mutations and overexpression of the tyrosine kinase Fibroblast Growth Factor Receptor 3 (FGFR3) are strongly associated with bladder cancer, evidence for their functional involvement in the pathogenesis remains elusive. Previously we showed that activation of Fgfr3 alone is not sufficient to initiate urothelial tumourigenesis in mice. Here we hypothesise that cooperating mutations are required for Fgfr3-dependent tumourigenesis in the urothelium and analyse a mouse model in which an inhibitor of Pi3k-Akt signalling, Pten, is deleted in concert with Fgfr3 activation (UroIICreFgfr3(+/) (K644E) Pten(flox) (/flox) ). Two main phonotypical characteristics observed in the urothelium were increased urothelial thickness and abnormal cellular histopathology, including vacuolisation, condensed cellular appearance, enlargement of cells and nuclei, and loss of polarity. These changes were not observed when either mutation was present individually. Expression patterns of known urothelial proteins indicated the abnormal cellular differentiation. Furthermore, quantitative analysis showed that Fgfr3 and Pten mutations cooperatively caused cellular enlargement, while Pten contributed to an increased cell proliferation. Finally, FGFR3 overexpression was analysed along the level of phosphorylated mTOR in sixty-six T1 urothelial tumours in tissue microarray, which supported the occurrence of functional association of these two signalling pathways in urothelial pathogenesis. Taken together, this study provides evidence supporting a functional role of FGFR3 in the process of pathogenesis in urothelial neoplasm. Given the wide availability of inhibitors specific to FGF signalling pathways, our model may open the avenue for FGFR3-targeted translation in urothelial disease.
The Journal of Pathology 02/2014; · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Autophagy has a major role in cellular homeostasis and has been implicated in various forms of human disease. The role of autophagy in cancer seems to be complex, with reports indicating both pro-tumorigenic and tumour-suppressive roles. Here we show, in a humanized genetically-modified mouse model of pancreatic ductal adenocarcinoma (PDAC), that autophagy's role in tumour development is intrinsically connected to the status of the tumour suppressor p53. Mice with pancreases containing an activated oncogenic allele of Kras (also called Ki-Ras)-the most common mutational event in PDAC-develop a small number of pre-cancerous lesions that stochastically develop into PDAC over time. However, mice also lacking the essential autophagy genes Atg5 or Atg7 accumulate low-grade, pre-malignant pancreatic intraepithelial neoplasia lesions, but progression to high-grade pancreatic intraepithelial neoplasias and PDAC is blocked. In marked contrast, in mice containing oncogenic Kras and lacking p53, loss of autophagy no longer blocks tumour progression, but actually accelerates tumour onset, with metabolic analysis revealing enhanced glucose uptake and enrichment of anabolic pathways, which can fuel tumour growth. These findings provide considerable insight into the role of autophagy in cancer and have important implications for autophagy inhibition in cancer therapy. In this regard, we also show that treatment of mice with the autophagy inhibitor hydroxychloroquine, which is currently being used in several clinical trials, significantly accelerates tumour formation in mice containing oncogenic Kras but lacking p53.
[Show abstract][Hide abstract] ABSTRACT: Regulation of metabolic pathways plays an important role in controlling cell growth, proliferation, and survival. TIGAR acts as a fructose-2,6-bisphosphatase, potentially promoting the pentose phosphate pathway to produce NADPH for antioxidant function and ribose-5-phosphate for nucleotide synthesis. The functions of TIGAR were dispensable for normal growth and development in mice but played a key role in allowing intestinal regeneration in vivo and in ex vivo cultures, where growth defects due to lack of TIGAR were rescued by ROS scavengers and nucleosides. In a mouse intestinal adenoma model, TIGAR deficiency decreased tumor burden and increased survival, while elevated expression of TIGAR in human colon tumors suggested that deregulated TIGAR supports cancer progression. Our study demonstrates the importance of TIGAR in regulating metabolism for regeneration and cancer development and identifies TIGAR as a potential therapeutic target in diseases such as ulcerative colitis and intestinal cancer.
[Show abstract][Hide abstract] ABSTRACT: The Adenomatous Polyposis Coli (APC) gene is mutated in the majority of colorectal cancers (CRCs). Loss of APC leads to constitutively active WNT signaling, hyperproliferation, and tumorigenesis. Identification of pathways that facilitate tumorigenesis after APC loss is important for therapeutic development. Here, we show that RAC1 is a critical mediator of tumorigenesis after APC loss. We find that RAC1 is required for expansion of the LGR5 intestinal stem cell (ISC) signature, progenitor hyperproliferation, and transformation. Mechanistically, RAC1-driven ROS and NF-κB signaling mediate these processes. Together, these data highlight that ROS production and NF-κB activation triggered by RAC1 are critical events in CRC initiation.
[Show abstract][Hide abstract] ABSTRACT: Tissue progenitor cells are an attractive target for regenerative therapy. In various organs, bone marrow cell (BMC) therapy has shown promising preliminary results, but to date no definite mechanism has been demonstrated to account for the observed benefit in organ regeneration. Tissue injury and regeneration is invariably accompanied by macrophage infiltration, but their influence upon the progenitor cells is incompletely understood, and direct signaling pathways may be obscured by the multiple roles of macrophages during organ injury. We therefore examined a model without injury; a single i.v. injection of unfractionated BMCs in healthy mice. This induced ductular reactions (DRs) in healthy mice. We demonstrate that macrophages within the unfractionated BMCs are responsible for the production of DRs, engrafting in the recipient liver and localizing to the DRs. Engrafted macrophages produce the cytokine TWEAK (TNF-like weak inducer of apoptosis) in situ. We go on to show that recombinant TWEAK activates DRs and that BMC mediated DRs are TWEAK dependent. DRs are accompanied by liver growth, occur in the absence of liver tissue injury and hepatic progenitor cells can be isolated from the livers of mice with DRs. Overall these results reveal a hitherto undescribed mechanism linking macrophage infiltration to DRs in the liver and highlight a rationale for macrophage derived cell therapy in regenerative medicine.
Proceedings of the National Academy of Sciences 04/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Within the intestinal epithelium, c-Myc has been characterized as a target of β-catenin-TCF signalling (He et al., Science 281:1509-1512, 1998). Given the most commonly mutated tumor suppressor gene within colorectal cancer (CRC) is the APC (Adenomatous Polyposis Coli) gene, a negative regulator of β-catenin-TCF signalling (Korinek et al., Science 275:1784-1787, 1997), loss of APC leads to Myc deregulation in the vast majority of CRC. This probably explains the numerous studies investigating c-Myc function within the intestinal epithelium. These have shown that c-Myc inhibition or deletion in the adult intestine results in proliferative defects (Muncan et al., Mol Cell Biol 26:8418-8426, 2006; Soucek et al., Nature 455:679-683, 2008). Importantly, intestinal enterocytes are able to survive in the absence of c-Myc which has allowed us (and others) to test the role of c-Myc in intestinal regeneration and tumorigenesis. Remarkably c-Myc deletion suppresses all the phenotypes of the Apc tumor suppressor gene loss and stops intestinal regeneration (Ashton et al., Dev Cell 19:259-269, 2010; Sansom et al., Oncogene 29:2585-2590, 2007). This suggests a clear therapeutic rationale for targeting c-Myc in CRC. Moreover haploinsufficiency for c-Myc in this tissue also reduces intestinal tumorigenesis (Athineos and Sansom, Oncogene 29:2585-2590, 2010; Yekkala and Baudino, Mol Cancer Res 5:1296-1303, 2007), and overexpression of c-Myc affects tissue homeostasis (Finch et al., Mol Cell Biol 29:5306-5315, 2009; Murphy et al., Cancer Cell 14:447-457, 2008).In this chapter we will provide an overview of our current laboratory protocols to characterize c-Myc function in intestinal homeostasis, regeneration, and tumorigenesis in vivo and in vitro.
[Show abstract][Hide abstract] ABSTRACT: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that integrates signals downstream of integrin and growth factor activation. Previously, we have shown that skin-specific loss of fak prevents chemically induced skin carcinogenesis in mice following phorbol ester treatment. In this study, we show that skin-specific deletion of fak prevents mobilization of stem cells within the bulge region of the hair follicle, which are the precursors of papillomas following phorbol ester treatment. We also show that phorbol ester treatment results in activation of-catenin within the skin and that FAK is required for β-catenin-induced stem cell mobilization. In addition, inhibition of Src kinase activity, a major binding partner of FAK also prevents stem cell mobilization. We show that FAK is required for the nuclear localization of β-catenin in the skin following phorbol ester treatment and the transcriptional activation of the β-catenin target gene c-Myc. This provides the first evidence of cross-talk between integrin and Wnt signalling pathways in the control of epidermal stem cells and the early events associated with skin carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: It is clear from epidemiological studies that excess iron is associated with increased risk of colorectal cancer; however, questions regarding the mechanism of how iron increases cancer risk, the source of the excess iron (circulating or luminal), and whether iron reduction represents a potential therapeutic option remain unanswered. In this study, we show that after Apc deletion, the cellular iron acquisition proteins TfR1 and DMT1 are rapidly induced. Conversely, restoration of APC reduces cellular iron due to repression of these proteins. To test the functional importance of these findings, we performed in vivo investigations of the impact of iron levels on intestinal tumorigenesis. Strikingly, depletion of luminal (but not systemic) iron strongly suppressed murine intestinal tumorigenesis, whereas increased luminal iron strongly promoted tumorigenesis. Taken together, our data definitively delineate iron as a potent modifier of intestinal tumorigenesis and have important implications for dietary iron supplementation in patients at high risk of colorectal cancer.
[Show abstract][Hide abstract] ABSTRACT: A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active β-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear β-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach.Oncogene advance online publication, 4 June 2012; doi:10.1038/onc.2012.224.
[Show abstract][Hide abstract] ABSTRACT: Colorectal cancers commonly carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein contributes to the stabilization of microtubules. Consistently, microtubules in cells lacking APC depolymerize more readily in response to microtubule-destabilizing drugs. This raises the possibility that such agents are suitable for treatment of APC-deficient cancers. However, APC-deficient cells have a compromised spindle assembly checkpoint, which renders them less sensitive to killing by microtubule poisons whose toxicity relies on the induction of prolonged mitotic arrest. Here, we describe the novel discovery that the clinically used microtubule-depolymerizing drug vinorelbine (Navelbine) kills APC-deficient cells in culture and in intestinal tissue more effectively than it kills wild-type cells. This is due to the ability of vinorelbine to kill cells in interphase independently of mitotic arrest. Consistent with a role for p53 in cell death in interphase, depletion of p53 renders cells less sensitive to vinorelbine, but only in the presence of wild-type APC. The pro-apoptotic protein BIM (also known as BCL2L11) is recruited to mitochondria in response to vinorelbine, where it can inhibit the anti-apoptotic protein BCL2, suggesting that BIM mediates vinorelbine-induced cell death. This recruitment of BIM is enhanced in cells lacking APC. Consistently, BIM depletion dampens the selective effect of vinorelbine on these cells. Our findings reveal that vinorelbine is a potential therapeutic agent for colorectal cancer, but they also illustrate the importance of the APC tumour suppressor status when predicting therapeutic efficacy.
[Show abstract][Hide abstract] ABSTRACT: During chronic injury a population of bipotent hepatic progenitor cells (HPCs) become activated to regenerate both cholangiocytes and hepatocytes. Here we show in human diseased liver and mouse models of the ductular reaction that Notch and Wnt signaling direct specification of HPCs via their interactions with activated myofibroblasts or macrophages. In particular, we found that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signaling in HPCs and thus their biliary specification to cholangiocytes. Alternatively, during hepatocyte regeneration, macrophage engulfment of hepatocyte debris induced Wnt3a expression. This resulted in canonical Wnt signaling in nearby HPCs, thus maintaining expression of Numb (a cell fate determinant) within these cells and the promotion of their specification to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted.
Nature medicine 03/2012; 18(4):572-9. · 27.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.
[Show abstract][Hide abstract] ABSTRACT: Although deregulation of the Wnt signalling pathway has been implicated in urothelial cell carcinoma (UCC), the functional significance is unknown. To test its importance, we have targeted expression of an activated form of β-catenin to the urothelium of transgenic mice using Cre-Lox technology (UroIICRE(+) β-catenin(exon3/+)). Expression of this activated form of β-catenin led to the formation of localized hyperproliferative lesions by 3 months, which did not progress to malignancy. These lesions were characterized by a marked increase of the phosphatase and tensin homologue (PTEN) tumour suppressor protein. This appears to be a direct consequence of activating Wnt signalling in the bladder as conditional deletion of the adenomatous polyposis coli (Apc) gene within the adult bladder led rapidly to coincident β-catenin and PTEN expression. This PTEN expression blocked proliferation. Next, we combined PTEN deficiency with β-catenin activation and found that this caused papillary UCC. These tumours had increased pAKT signalling and were dependent on mammalian target of rapamycin (mTOR). Importantly, in human UCC, there was a significant correlation between high levels of β-catenin and pAKT (and low levels of PTEN). Taken together these data show that deregulated Wnt signalling has a critical role in promoting UCC, and suggests that human UCC that have high levels of Wnt and PI3 kinase signalling may be responsive to mTOR inhibition.