Ultrasound mediated delivery of quantum dots from a proof of concept capsule endoscope to the gastrointestinal wall.

Biologic drugs, defined as therapeutic agents produced from or containing components of a living organism, are of growing importance to the pharmaceutical industry. Though oral delivery of medicine is convenient, biologics require invasive injections because of their poor bioavailability via oral routes. Delivery of biologics to the small intestine using electronic delivery with devices that are similar to capsule endoscopes is a promising means of overcoming this limitation and does not require reformulation of the therapeutic agent. The efficacy of such capsule devices for drug delivery could be further improved by increasing the permeability of the intestinal tract lining with an integrated ultrasound transducer to increase uptake. This paper describes a novel proof of concept capsule device capable of electronic application of focused ultrasound and delivery of therapeutic agents. Fluorescent markers, which were chosen as a model drug, were used to demonstrate in vivo delivery in the porcine small intestine with this capsule. We show that the fluorescent markers can penetrate the mucus layer of the small intestine at low acoustic powers when combining microbubbles with focused ultrasound during in vivo experiments using porcine models. This study illustrates how such a device could be potentially used for gastrointestinal drug delivery and the challenges to be overcome before focused ultrasound and microbubbles could be used with this device for the oral delivery of biologic therapeutics.

A Learning-Based Microultrasound System for the Detection of Inflammation of the Gastrointestinal Tract.

Inflammation of the gastrointestinal (GI) tract accompanies several diseases, including Crohn's disease. Currently, video capsule endoscopy and deep bowel enteroscopy are the main means for direct visualisation of the bowel surface. However, the use of optical imaging limits visualisation to the luminal surface only, which makes early-stage diagnosis difficult. In this study, we propose a learning enabled microultrasound ( µ US) system that aims to classify inflamed and non-inflamed bowel tissues. µ US images of the caecum, small bowel and colon were obtained from mice treated with agents to induce inflammation. Those images were then used to train three deep learning networks and to provide a ground truth of inflammation status. The classification accuracy was evaluated using 10-fold evaluation and additional B-scan images. Our deep learning approach allowed robust differentiation between healthy tissue and tissue with early signs of inflammation that is not detectable by current endoscopic methods or by human inspection of the µ US images. The methods may be a foundation for future early GI disease diagnosis and enhanced management with computer-aided imaging.

Loss of adenomatous polyposis coli function renders intestinal epithelial cells resistant to the cytokine IL-22.

Interleukin-22 (IL-22) is a critical immune defence cytokine that maintains intestinal homeostasis and promotes wound healing and tissue regeneration, which can support the growth of colorectal tumours. Mutations in the adenomatous polyposis coli gene (Apc) are a major driver of familial colorectal cancers (CRCs). How IL-22 contributes to APC-mediated tumorigenesis is poorly understood. To investigate IL-22 signalling in wild-type (WT) and APC-mutant cells, we performed RNA sequencing (RNAseq) of IL-22-treated murine small intestinal epithelial organoids. In WT epithelia, antimicrobial defence and cellular stress response pathways were most strongly induced by IL-22. Surprisingly, although IL-22 activates signal transducer and activator of transcription 3 (STAT3) in APC-mutant cells, STAT3 target genes were not induced. Our analyses revealed that ApcMin/Min cells are resistant to IL-22 due to reduced expression of the IL-22 receptor, and increased expression of inhibitors of STAT3, particularly histone deacetylases (HDACs). We further show that IL-22 increases DNA damage and genomic instability, which can accelerate cellular transition from heterozygosity (ApcMin/+) to homozygosity (ApcMin/Min) to drive tumour formation. Our data reveal an unexpected role for IL-22 in promoting early tumorigenesis while excluding a function for IL-22 in transformed epithelial cells.

Identification of Endogenous Adenomatous Polyposis Coli Interaction Partners and ß-Catenin-Independent Targets by Proteomics.

Adenomatous Polyposis Coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the Wnt signaling pathway by promoting the degradation of ß-catenin, but the extent to which APC exerts Wnt/ß-catenin-independent tumor-suppressive activity is unclear. To identify interaction partners and ß-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed tandem mass tag-based mass spectrometry. Affinity enrichment-mass spectrometry identified more than 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of ß-catenin. Combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an APC-containing destruction complex. We validated the interaction between endogenous MINK1 and APC and further confirmed the negative, and ß-catenin-independent, regulation of MINK1 by APC. Increased Mink1/Msn levels were also observed in mouse intestinal tissue and Drosophila follicular cells expressing mutant Apc/APC when compared with wild-type tissue/cells. Collectively, our results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease. IMPLICATIONS: The tumor-suppressive function of APC, the most frequently mutated gene in colorectal cancer, is mainly attributed to its role in ß-catenin/Wnt signaling. Our study substantially expands the list of APC interaction partners and reveals that approximately half of the changes in the cellular proteome induced by loss of APC function are mediated by ß-catenin-independent mechanisms.

Lgr5+ intestinal stem cells reside in an unlicensed G1 phase.

During late mitosis and the early G1 phase, the origins of replication are licensed by binding to double hexamers of MCM2-7. In this study, we investigated how licensing and proliferative commitment are coupled in the epithelium of the small intestine. We developed a method for identifying cells in intact tissue containing DNA-bound MCM2-7. Interphase cells above the transit-amplifying compartment had no DNA-bound MCM2-7, but still expressed the MCM2-7 protein, suggesting that licensing is inhibited immediately upon differentiation. Strikingly, we found most proliferative Lgr5+ stem cells are in an unlicensed state. This suggests that the elongated cell-cycle of intestinal stem cells is caused by an increased G1 length, characterized by dormant periods with unlicensed origins. Significantly, the unlicensed state is lost in Apc-mutant epithelium, which lacks a functional restriction point, causing licensing immediately upon G1 entry. We propose that the unlicensed G1 phase of intestinal stem cells creates a temporal window when proliferative fate decisions can be made.

Interkinetic nuclear migration and basal tethering facilitates post-mitotic daughter separation in intestinal organoids.

Homeostasis of renewing tissues requires balanced proliferation, differentiation and movement. This is particularly important in the intestinal epithelium where lineage tracing suggests that stochastic differentiation choices are intricately coupled to the position of a cell relative to a niche. To determine how position is achieved, we followed proliferating cells in intestinal organoids and discovered that the behaviour of mitotic sisters predicted long-term positioning. We found that, normally, 70% of sisters remain neighbours, while 30% lose contact and separate after cytokinesis. These post-mitotic placements predict longer term differences in positions assumed by sisters: adjacent sisters reach similar positions over time; in a pair of separating sisters, one remains close to its birthplace while the other is displaced upward. Computationally modelling crypt dynamics confirmed that post-mitotic separation leads to sisters reaching different compartments. We show that interkinetic nuclear migration, cell size and asymmetric tethering by a process extending from the basal side of cells contribute to separations. These processes are altered in adenomatous polyposis coli (Apc) mutant epithelia where separation is lost. We conclude that post-mitotic placement contributes to stochastic niche exit and, when defective, supports the clonal expansion of Apc mutant cells.

Acoustic Sensing and Ultrasonic Drug Delivery in Multimodal Theranostic Capsule Endoscopy.

Video capsule endoscopy (VCE) is now a clinically accepted diagnostic modality in which miniaturized technology, an on-board power supply and wireless telemetry stand as technological foundations for other capsule endoscopy (CE) devices. However, VCE does not provide therapeutic functionality, and research towards therapeutic CE (TCE) has been limited. In this paper, a route towards viable TCE is proposed, based on multiple CE devices including important acoustic sensing and drug delivery components. In this approach, an initial multimodal diagnostic device with high-frequency quantitative microultrasound that complements video imaging allows surface and subsurface visualization and computer-assisted diagnosis. Using focused ultrasound (US) to mark sites of pathology with exogenous fluorescent agents permits follow-up with another device to provide therapy. This is based on an US-mediated targeted drug delivery system with fluorescence imaging guidance. An additional device may then be utilized for treatment verification and monitoring, exploiting the minimally invasive nature of CE. While such a theranostic patient pathway for gastrointestinal treatment is presently incomplete, the description in this paper of previous research and work under way to realize further components for the proposed pathway suggests it is feasible and provides a framework around which to structure further work.

Ultrasound capsule endoscopy: sounding out the future.

Video capsule endoscopy (VCE) has been of immense benefit in the diagnosis and management of gastrointestinal (GI) disorders since its introduction in 2001. However, it suffers from a number of well recognized deficiencies. Amongst these is the limited capability of white light imaging, which is restricted to analysis of the mucosal surface. Current capsule endoscopes are dependent on visual manifestation of disease and limited in regards to transmural imaging and detection of deeper pathology. Ultrasound capsule endoscopy (USCE) has the potential to overcome surface only imaging and provide transmural scans of the GI tract. The integration of high frequency microultrasound (µUS) into capsule endoscopy would allow high resolution transmural images and provide a means of both qualitative and quantitative assessment of the bowel wall. Quantitative ultrasound (QUS) can provide data in an objective and measurable manner, potentially reducing lengthy interpretation times by incorporation into an automated diagnostic process. The research described here is focused on the development of USCE and other complementary diagnostic and therapeutic modalities. Presently investigations have entered a preclinical phase with laboratory investigations running concurrently.

Paneth Cell-Rich Regions Separated by a Cluster of Lgr5+ Cells Initiate Crypt Fission in the Intestinal Stem Cell Niche.

The crypts of the intestinal epithelium house the stem cells that ensure the continual renewal of the epithelial cells that line the intestinal tract. Crypt number increases by a process called crypt fission, the division of a single crypt into two daughter crypts. Fission drives normal tissue growth and maintenance. Correspondingly, it becomes less frequent in adulthood. Importantly, fission is reactivated to drive adenoma growth. The mechanisms governing fission are poorly understood. However, only by knowing how normal fission operates can cancer-associated changes be elucidated. We studied normal fission in tissue in three dimensions using high-resolution imaging and used intestinal organoids to identify underlying mechanisms. We discovered that both the number and relative position of Paneth cells and Lgr5+ cells are important for fission. Furthermore, the higher stiffness and increased adhesion of Paneth cells are involved in determining the site of fission. Formation of a cluster of Lgr5+ cells between at least two Paneth-cell-rich domains establishes the site for the upward invagination that initiates fission.

Dichlorodiphenyldichloroethylene exposure reduces r-GCS via suppressed Nrf2 in HepG2 cells.

p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), the major isomer of persistent 1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethane metabolite, is highly associated with the risk of liver cancer. γ-glutamyl-cysteine synthetase (γ-GCS), which is the rate-limiting enzyme of glutathione (GSH) biosynthesis and an important scavenger of reactive oxygen species (ROS), is considered as a potential therapeutic target for many cancers. However, the association between the body burden of p,p'-DDE and γ-GCS has not been fully established. Here, we indicated that low doses of p,p'-DDE exposure promoted the proliferation and decreased γ-GCS activity of HepG2 cells in a dose- and time-dependent manner. In addition, p,p'-DDE elevated ROS content and attenuated glutathione peroxidase activity. This was accompanied with inhibitions of NF-E2-related factor 2 (Nrf2) at the mRNA and protein levels. ROS inhibitor supplement could significantly reverse these effects. Moreover, the addition of the proteasome inhibitor, MG132, strongly reversed the p,p'-DDE-reduced Nrf2 expression and γ-GCS activity. Consistently, GSH content was in line with the alteration of γ-GCS. Collectively, the results indicate that p,p'-DDE treatment downregulates γ-GCS activity in HepG2 cells by inducing ROS-mediated Nrf2 loss.

GRP78 is implicated in the modulation of tumor aerobic glycolysis by promoting autophagic degradation of IKKß.

Compared with normal differentiated cells, cancer cells take up much more glucose and metabolize it mainly via aerobic glycolysis. This metabolic phenotype is characterized with high expression of glucose transporters (Gluts) and pyruvate kinase M2 (PKM2). Glucose regulated protein 78 (GRP78) is a glucose-sensing protein and frequently up-regulated in cancer cells, however, whether it is directly implicated in glucose metabolism remains to be elucidated. Here we report that upon glucose deficiency, the induction of GRP78 resulted in enhanced HIF-1α transcription, accompanied by a transient increased expression of Glut-1. In addition, GRP78 was likely to facilitate the membrane translocation of Glut-1 via protein-protein interaction. Glucose starvation-stimulated GRP78 also impaired the expression of PKM2 but promoted the expression of mitochondrial pyruvate dehydrogenase A (PDHA) and B (PDHB), resulting in the metabolic shift from glycolysis to the TCA cycle. Interestingly, the inhibition of PKM2 by GRP78 was abrogated when glucose supply was restored, suggesting that GRP78 and PKM2 expressions are adaptable to the nutritional levels in the microenvironment. Further mechanistic study indicated that GRP78 overexpression activated the Class III PI3K-mediated autophagy pathway and induced autophagic degradation of IKKß, which caused inactivation of NF-κB pathway and subsequently altered the expression of PKM2 and HIF-1α. Our study establishes GRP78 and PKM2 as the crucial molecular links between cancer cell glucose metabolism and tumor microenvironment alterations.

Expression, purification and renaturation of truncated human integrin ß1 from inclusion bodies of Escherichia coli.

Integrins are a family of transmembrane receptors and among their members, integrin ß1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin ß1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin ß1. The recombinant plasmid was transformed into Escherichiacoli BL21 (DE3) cells and after induction by isopropyl-ß-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg/L of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS-PAGE. The integrity of the protein was shown by Western blot using monoclonal antibodies against his-tag and integrin ß1. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn(2+) stimulation.

N-terminal truncation mutations of adenomatous polyposis coli are associated with primary cilia defects.

Adenomatous polyposis coli (APC) gene is a tumor suppressor gene and its truncated mutations cause a few cilia-related diseases such as Gardner's syndrome. However, little is known about the mechanism that links APC mutations and cilia disorder. APC mutations lead to the expression of N-terminal fragments, which have dominant effects in tumors owing to loss of the C-terminal region or a gain of function. The present study investigated the impact of tumor-associated N-terminal APC fragments on primary cilia assembly and the possible molecular mechanism involved. We discovered that expression of tumor-associated N-terminal APC fragments (APC-N, APC-N1, APC-N2, and APC-N3, which contain amino acids 1-1018, 1-448, 449-781, and 782-1018 respectively), resulted in primary cilia defects. We found that a ß-catenin/PI3K/AKT/GSK-3ß feedback signal cascade is responsible for causing N-terminal APC fragment-induced cilia defects. In this cascade, dysfunctions of both ß-catenin and GSK-3ß were involved in the up-regulation of HDAC6 and subsequent decreased acetylated tubulin levels, which thereby led to cilia defects. These data suggest a mechanism for linking N-terminal APC fragments and cilia loss, thus accelerating our understanding of human cilia-related diseases such as Gardner's syndrome and their cause due to APC mutations.

Direct contacts with colon cancer cells regulate the differentiation of bone marrow mesenchymal stem cells into tumor associated fibroblasts.

Tumor-stroma interactions are referred to as essential events in tumor progression. There has been growing attention that bone marrow-derived mesenchymal stem cells (BMSCs) can travel to tumor stroma, where they differentiate into tumor-associated fibroblast (TAF)-like cells, a predominant tumor-promoting stromal cell. However, little is definitively known about the contributors for this transition. Here, using an in vitro direct co-culture model of colon cancer cells and BMSCs, we identify that colon cancer cells can induce adjoining BMSCs to exhibit the typical characteristic of TAFs, with increased expression of α-smooth muscle actin (α-SMA). Importantly, the present data also reveals that activated Notch signaling mediates transformation of BMSCs to TAFs through the downstream TGF-ß/Smad signaling pathway.

The organochlorine p,p'-dichlorodiphenyltrichloroethane induces colorectal cancer growth through Wnt/ß-catenin signaling.

Dichlorodiphenyltrichloroethane (DDT), an organochlorine pollutant, is associated with several types of cancer. However, the relationship between DDT and colorectal cancer is uncertain. In this study, the impact of p,p'-DDT on colorectal cancer growth was evaluated using both in vitro and in vivo models. Our results indicated that the proliferation of human colorectal adenocarcinoma DLD1 cells was significantly promoted after exposed to low concentrations of p,p'-DDT ranging from 10(-12) to 10(-7) M for 96 h. Exposure to p,p'-DDT from 10(-10) to 10(-8) M led to upregulation of phospho-GSK3ß (Ser9), ß-catenin, c-Myc and cyclin D1 in DLD1 cells. RNA interference of ß-catenin inhibited the proliferation of DLD1 cells stimulated by p,p'-DDT. Inhibiting of estrogen receptors (ERs) had no significant effect on the action of p,p'-DDT. Treatment with p,p'-DDT induced production of intracellular reactive oxygen species (ROS) and inhibited superoxide dismutase (SOD) activity in DLD1 cells. Treatment with N-acetyl-L-cysteine (NAC), a ROS inhibitor, suppressed the induction of Wnt/ß-catenin signaling and DLD1 cell proliferation by p,p'-DDT. Moreover, in a mouse xenograft model, 5 nmol/kg p,p'-DDT resulted in increased tumor size, oxidative stress and Wnt/ß-catenin signaling. These results indicated that low concentrations of p,p'-DDT promoted colorectal cancer growth through Wnt/ß-catenin signaling, which was mediated by oxidative stress. The finding suggests an association between low concentrations of p,p'-DDT exposure and colorectal cancer progression.

A novel protein extracted from foxtail millet bran displays anti-carcinogenic effects in human colon cancer cells.

Millet is an important cereal food and exhibits multiple biological activities, including immunodulatory, antioxidant, antifungal and anti-hyperglycemia effects. Herein, we describe a novel 35kDa protein with anti-cancer properties, named FMBP, which was extracted and purified from foxtail millet bran by cell-based screening. FMBP is highly homologous to peroxidase as revealed by mass spectrometry and gene sequencing analysis. Furthermore, in vivo anti-tumor results implicated that the novel protein FMBP had the ability to suppress xenografted tumor growth in nude mice. Mechanistically, FMBP is able to suppress colon cancer cell growth through induction of G1 phase arrest and also causes a loss of mitochondrial transmembrane potential which results in caspase-dependent apoptosis in colon cancer cells. Notably, FMBP has much lower toxicity in normal colon epithelial cells. Taken together, FMBP has targeted anti-colon cancer effects and may serve as a therapeutic agent against colon cancer.

The adenomatous polyposis coli protein contributes to normal compaction of mitotic chromatin.

The tumour suppressor Adenomatous Polyposis Coli (APC) is required for proper mitosis; however, the exact role of APC in mitosis is not understood. Using demembranated sperm chromatin exposed to meiotic Xenopus egg extract and HeLa cells expressing fluorescently labelled histones, we established that APC contributes to chromatin compaction. Sperm chromatin in APC-depleted Xenopus egg extract frequently formed tight round or elongated structures. Such abnormally compacted chromatin predominantly formed spindles with low microtubule content. Furthermore, in mitotic HeLa cells expressing GFP- and mCherry-labelled H2B histones, depletion of APC caused a decrease in the donor fluorescence lifetime of neighbouring fluorophores, indicative of excessive chromatin compaction. Profiling the chromatin-associated proteome of sperm chromatin incubated with Xenopus egg extracts revealed temporal APC-dependent changes in the abundance of histones, closely mirrored by chromatin-associated Topoisomerase IIa, condensin I complex and Kif4. In the absence of APC these factors initially accumulated on chromatin, but then decreased faster than in controls. We also found and validated significant APC-dependent changes in chromatin modifiers Set-a and Rbbp7. Both were decreased on chromatin in APC-depleted extract; in addition, the kinetics of association of Set-a with chromatin was altered in the absence of APC.

Tumorigenic fragments of APC cause dominant defects in directional cell migration in multiple model systems.

Nonsense mutations that result in the expression of truncated, N-terminal, fragments of the adenomatous polyposis coli (APC) tumour suppressor protein are found in most sporadic and some hereditary colorectal cancers. These mutations can cause tumorigenesis by eliminating ß-catenin-binding sites from APC, which leads to upregulation of ß-catenin and thereby results in the induction of oncogenes such as MYC. Here we show that, in three distinct experimental model systems, expression of an N-terminal fragment of APC (N-APC) results in loss of directionality, but not speed, of cell motility independently of changes in ß-catenin regulation. We developed a system to culture and fluorescently label live pieces of gut tissue to record high-resolution three-dimensional time-lapse movies of cells in situ. This revealed an unexpected complexity of normal gut cell migration, a key process in gut epithelial maintenance, with cells moving with spatial and temporal discontinuity. Quantitative comparison of gut tissue from wild-type mice and APC heterozygotes (APC(Min/+); multiple intestinal neoplasia model) demonstrated that cells in precancerous epithelia lack directional preference when moving along the crypt-villus axis. This effect was reproduced in diverse experimental systems: in developing chicken embryos, mesoderm cells expressing N-APC failed to migrate normally; in amoeboid Dictyostelium, which lack endogenous APC, expressing an N-APC fragment maintained cell motility, but the cells failed to perform directional chemotaxis; and multicellular Dictyostelium slug aggregates similarly failed to perform phototaxis. We propose that N-terminal fragments of APC represent a gain-of-function mutation that causes cells within tissue to fail to migrate directionally in response to relevant guidance cues. Consistent with this idea, crypts in histologically normal tissues of APC(Min/+) intestines are overpopulated with cells, suggesting that a lack of migration might cause cell accumulation in a precancerous state.

The microtubule poison vinorelbine kills cells independently of mitotic arrest and targets cells lacking the APC tumour suppressor more effectively.

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.

Adenomatous polyposis coli and hypoxia-inducible factor-1{alpha} have an antagonistic connection.

The tumor suppressor adenomatous polyposis coli (APC) is mutated in the majority of colorectal cancers and is best known for its role as a scaffold in a Wnt-regulated protein complex that determines the availability of ß-catenin. Another common feature of solid tumors is the presence of hypoxia as indicated by the up-regulation of hypoxia-inducible factors (HIFs) such as HIF-1α. Here, we demonstrate a novel link between APC and hypoxia and show that APC and HIF-1α antagonize each other. Hypoxia results in reduced levels of APC mRNA and protein via a HIF-1α-dependent mechanism. HIF-1α represses the APC gene via a functional hypoxia-responsive element on the APC promoter. In contrast, APC-mediated repression of HIF-1α requires wild-type APC, low levels of ß-catenin, and nuclear factor-κB activity. These results reveal down-regulation of APC as a new mechanism that contributes to the survival advantage induced by hypoxia and also show that loss of APC mutations produces a survival advantage by mimicking hypoxic conditions.