Nuclear partitioning of Prohibitin 1 inhibits Wnt/β-catenin-dependent intestinal tumorigenesis.
ABSTRACT: The Wnt/β-catenin signaling pathway is aberrantly activated in the majority of colorectal cancer cases due to somatic mutations in the adenomatous polyposis coli (APC) gene. Prohibitin 1 (PHB1) serves pleiotropic cellular functions with dynamic subcellular trafficking, facilitating signaling crosstalk between organelles. Nuclear-localized PHB1 is an important regulator of gene transcription. Using mice with inducible intestinal epithelial cell (IEC)-specific deletion of Phb1 (Phb1iΔIEC) and mice with IEC-specific overexpression of Phb1 (Phb1Tg), we demonstrate that IEC-specific PHB1 combats intestinal tumorigenesis in the ApcMin/+ mouse model by inhibiting Wnt/β-catenin signaling. Forced nuclear accumulation of PHB1 in human RKO or SW48 CRC cell lines increased AXIN1 expression and decreased cell viability. PHB1 deficiency in CRC cells decreased AXIN1 expression and increased β-catenin activation that was abolished by XAV939, a pharmacological AXIN stabilizer. These results define a role of PHB1 in inhibiting the Wnt/β-catenin pathway to influence the development of intestinal tumorigenesis. Induction of nuclear PHB1 trafficking provides a novel therapeutic option to influence AXIN1 expression and the β-catenin destruction complex in Wnt-driven intestinal tumorigenesis.
Project description:Colorectal cancer exhibits aberrant activation of Wnt/?-catenin signaling. Many inhibitors of the Wnt/?-catenin pathway have been tested for Wnt-dependent cancers including colorectal cancer, but are unsuccessful due to severe adverse reactions. FL3 is a synthetic derivative of natural products called flavaglines, which exhibit anti-inflammatory and cytoprotective properties in intestinal epithelial cells, but has not been previously tested in cell or preclinical models of intestinal tumorigenesis. <i>In vitro</i> studies suggest that flavaglines target prohibitin 1 (PHB1) as a ligand, but this has not been established in the intestine. PHB1 is a highly conserved protein with diverse functions that depend on its posttranslational modifications and subcellular localization. Here, we demonstrate that FL3 combats intestinal tumorigenesis in the azoxymethane-dextran sodium sulfate and <i>Apc<sup>Min/+</sup></i> mouse models and in human colorectal cancer tumor organoids (tumoroids) by inhibiting Wnt/?-catenin signaling via induction of Axin1 expression. FL3 exhibited no change in cell viability in normal intestinal epithelial cells or human matched-normal colonoids. FL3 response was diminished in colorectal cancer cell lines and human colorectal cancer tumoroids harboring a mutation at S45 of ?-catenin. PHB1 deficiency in mice or in human colorectal cancer tumoroids abolished FL3-induced expression of Axin1 and drove tumoroid death. In colorectal cancer cells, FL3 treatment blocked phosphorylation of PHB1 at Thr258, resulting in its nuclear translocation and binding to the Axin1 promoter. These results suggest that FL3 inhibits Wnt/?-catenin signaling via PHB1-dependent activation of Axin1. FL3, therefore, represents a novel compound that combats Wnt pathway-dependent cancers, such as colorectal cancer. SIGNIFICANCE: Targeting of PHB1 by FL3 provides a novel mechanism to combat Wnt-driven cancers, with limited intestinal toxicity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3519/F1.large.jpg.
Project description:Objective- The Wnt/β-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/β-catenin signaling by induced overexpression of Axin1, an inhibitor of β-catenin signaling, specifically in endothelial cells ( Axin1 <sup>iEC</sup>- <sup>OE</sup>). AOE (Axin1 overexpression) in Axin1 <sup>iEC</sup>- <sup>OE</sup> mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/β-catenin driven CNS vascular development in zebrafish also suggested that Axin1 <sup>iEC</sup>- <sup>OE</sup> led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, β-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific β-catenin-responsive ECM signature was also repressed in Axin1 <sup>iEC</sup>- <sup>OE</sup> and endothelial cell-specific β-catenin-knockout mice ( Ctnnb1-KO<sup>iEC</sup>) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/β-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-β-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.
Project description:Intestinal epithelial self-renewal is tightly regulated by signaling pathways controlling stem cell proliferation, determination and differentiation. In particular, Wnt/β-catenin signaling controls intestinal crypt cell division, survival and maintenance of the stem cell niche. Most colorectal cancers are initiated by mutations activating the Wnt/β-catenin pathway. Wnt signals are transduced through Frizzled receptors and LRP5/LRP6 coreceptors to downregulate GSK3β activity, resulting in increased nuclear β-catenin. Herein, we explored if LRP6 expression is required for maintenance of intestinal homeostasis, regeneration and oncogenesis. Mice with an intestinal epithelial cell-specific deletion of <i>Lrp6</i> (<i>Lrp6</i><sup>IEC-KO</sup>) were generated and their phenotype analyzed. No difference in intestinal architecture nor in proliferative and stem cell numbers was found in <i>Lrp6</i><sup>IEC-KO</sup> mice in comparison to controls. Nevertheless, using ex vivo intestinal organoid cultures, we found that LRP6 expression was critical for crypt cell proliferation and stem cell maintenance. When exposed to dextran sodium sulfate, <i>Lrp6</i><sup>IEC-KO</sup> mice developed more severe colitis than control mice. However, loss of LRP6 did not affect tumorigenesis in <i>Apc</i><sup>Min/+</sup> mice nor growth of human colorectal cancer cells. By contrast, <i>Lrp6</i> silencing diminished anchorage-independent growth of <i>BRaf</i><sup>V600E</sup>-transformed intestinal epithelial cells (IEC). Thus, LRP6 controls intestinal stem cell functionality and is necessary for BRAF-induced IEC oncogenesis.
Project description:The Wnt/β-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by β-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein β-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human β-catenin destruction complex from purified components, recapitulating complex assembly, β-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote β-catenin capture, phosphorylation, and ubiquitylation. APC facilitates β-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCF<sup>β-TrCP</sup> E3 ligase complex in a β-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes β-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/β-catenin signaling.
Project description:Up to 60% of gastro-oesophageal junction (GEJ) adenocarcinomas show nuclear beta-catenin expression, pointing to activated T-cell factor (TCF)/beta-catenin-driven gene transcription. We demonstrate in five human GEJ adenocarcinoma cell lines that nuclear beta-catenin expression indeed correlates with enhanced TCF-mediated transcription of a reporter gene. In several tumour types, TCF/beta-catenin activation is caused by mutations in either adenomatous polyposis coli (APC), beta-catenin exon 3, AXIN1, AXIN2 or beta-transducin repeat-containing protein (beta-TrCP). In GEJ adenocarcinomas, very few APC and beta-catenin mutations have been found. Therefore, the mechanism of Wnt pathway activation remains unclear. In the present study, we did not find AXIN1 gene mutations in 17 GEJ tumours with nuclear beta-catenin expression (without beta-catenin exon 3 mutations). Six intragenic single nucleotide polymorphisms (SNPs) were identified. One of these, the AXIN1 gene T1942C SNP, has a frequency of 21% but is only very recently described despite numerous AXIN1 gene mutational studies. We provide evidence why this SNP was missed in single strand conformation polymorphism analyses. The AXIN1 gene G2063A variation was previously described as a gene mutation but we demonstrate that this is a polymorphism. With these six SNPs loss of heterozygosity (LOH) was found in 11 of 15 (73%) informative tumours. To investigate a possible AXIN1 gene dosage effect in GEJ tumours expressing nuclear beta-catenin, AXIN1 locus LOH was determined in 20 tumours expressing membranous and no nuclear beta-catenin. LOH was found in 10 of 13 (77%) informative cases. AXIN1 protein immunohistochemistry revealed cytoplasmic expression in all tumours irrespective of the presence of AXIN1 locus LOH. These data indicate that nuclear beta-catenin expression is indicative for activated Wnt signalling and that neither AXIN1 gene mutations nor AXIN1 locus LOH are involved in Wnt pathway activation in GEJ adenocarcinomas.
Project description:Signal transducer and activator of transcription 3 (Stat3) has been shown to play a role in intestinal regeneration and colitis-associated colon carcinogenesis. However, the role of Stat3 in the Wnt-driven sporadic intestinal tumorigenesis remains poorly understood. We examined the roles of Stat3 in intestinal regeneration and tumorigenesis by organoid culture experiments using Stat3<sup>∆IEC</sup> mouse-derived intestinal epithelial cells in which Stat3 was disrupted. The regeneration of intestinal mucosa and organoid formation were significantly suppressed by Stat3 disruption, which was compensated by Wnt activation. Furthermore, once organoids were recovered, Stat3 was no longer required for organoid growth. These results indicate that Stat3 and Wnt signaling cooperatively protect epithelial cells at the early phase of intestinal regeneration. In contrast, intestinal tumorigenesis was not suppressed by Stat3 disruption in adenomatous polyposis coli ( Apc) <sup>Δ716</sup> and Apc<sup>∆716</sup> Tgfbr2<sup>∆IEC</sup> mice, thus indicating that Stat3 is not required for Wnt activation-driven intestinal tumorigenesis. Mechanistically, Itga5 and Itga6 were down-regulated by Stat3 disruption, and focal adhesion kinase (FAK) activation was also suppressed. Notably, FAK inhibitor suppressed the organoid formation of wild-type epithelial cells. These results indicate that Stat3 is indispensable for the survival of epithelial cells through the activation of integrin signaling and the downstream FAK pathway; however, it is not required for the Wnt signaling-activated normal or tumor epithelial cells.-Oshima, H., Kok, S.-Y., Nakayama, M., Murakami, K., Voon, D. C.-C., Kimura, T., Oshima, M. Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.
Project description:Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/beta-catenin pathway, we challenged the allele combinations by genetically restricting intracellular beta-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/beta-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/beta-catenin signaling capacity similar to that in the germline of the Apc(min) mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc(min) mice arise independently of intestinal tumorigenesis. Together, the present genotype-phenotype analysis suggests tissue-specific response levels for the Wnt/beta-catenin pathway that regulate both physiological and pathophysiological conditions.
Project description:Casitas B-lineage lymphoma (c-Cbl) is a recently identified ubiquitin ligase of nuclear ?-catenin and a suppressor of colorectal cancer (CRC) growth in cell culture and mouse tumor xenografts. We hypothesized that reduction in c-Cbl in colonic epithelium is likely to increase the levels of nuclear ?-catenin in the intestinal crypt, augmenting CRC tumorigenesis in an adenomatous polyposis coli (APC<sup>?14/+</sup>) mouse model. Haploinsufficient c-Cbl mice (APC<sup>?14/+</sup> c-Cbl<sup>+/-</sup>) displayed a significant (threefold) increase in atypical hyperplasia and adenocarcinomas in the small and large intestines; however, no differences were noted in the adenoma frequency. In contrast to the APC<sup>?14/+</sup> c-Cbl<sup>+/+</sup> mice, APC<sup>?14/+</sup> c-Cbl<sup>+/-</sup> crypts showed nuclear ?-catenin throughout the length of the crypts and up-regulation of Axin2, a canonical Wnt target gene, and SRY-box transcription factor 9, a marker of intestinal stem cells. In contrast, haploinsufficiency of c-Cbl<sup>+/-</sup> alone was insufficient to induce tumorigenesis regardless of an increase in the number of intestinal epithelial cells with nuclear ?-catenin and SRY-box transcription factor 9 in APC<sup>+/+</sup> c-Cbl<sup>+/-</sup> mice. This study demonstrates that haploinsufficiency of c-Cbl results in Wnt hyperactivation in intestinal crypts and accelerates CRC progression to adenocarcinoma in the milieu of APC<sup>?14/+</sup>, a phenomenon not found with wild-type APC. While emphasizing the role of APC as a gatekeeper in CRC, this study also demonstrates that combined partial loss of c-Cbl and inactivation of APC significantly contribute to CRC tumorigenesis.
Project description:Metaplastic carcinomas are distinct invasive breast carcinomas with aberrant nonglandular differentiation, which may be spindle, squamous, or chondroid. The limited effective treatments result from the lack of knowledge of its molecular etiology. Given the role of the Wnt pathway in cell fate and in the development of breast cancer, we hypothesized that defects in this pathway may contribute to the development of metaplastic carcinomas.In 36 primary metaplastic carcinomas, we comprehensively determined the prevalence of and mechanism underlying beta-catenin and Wnt pathway deregulation using immunohistochemistry for beta-catenin expression and localization and mutational analysis for CTNNB1 (encoding beta-catenin), APC, WISP3, AXIN1, and AXIN2 genes. By immunohistochemistry, normal beta-catenin was seen as membrane staining, and it was aberrant when >5% of tumor cells had nuclear or cytoplasmic accumulation or reduced membrane staining.By immunohistochemistry, aberrant beta-catenin was present in 33 of 36 (92%) cases, revealing deregulation of the Wnt pathway. CTNNB1 missense mutations were detected in 7 of 27 (25.9%) tumors available for mutation analyses. All mutations affected the NH(2)-terminal domain of beta-catenin, presumably rendering the mutant protein resistant to degradation. Two of 27 (7.4%) tumors had mutations of APC, and 5 (18.5%) carried a frame shift mutation of WISP3. No AXIN1 or AXIN2 mutations were found.Activation of the Wnt signaling pathway is common in this specific subtype of breast carcinoma. The discovery of CTNNB1, APC, and WISP3 mutations may result in new treatments for patients with metaplastic carcinomas of the breast.
Project description:Colon carcinogenesis is a multiple-step process involving the accumulation of a series of genetic and epigenetic alterations. The most commonly initiating event of intestinal carcinogenesis is mutation of the adenomatous polyposis coli (APC) gene, which leads to activation of the Wnt/?-catenin pathway. Olfactomedin 4 (OLFM4) has emerged as an intestinal stem-cell marker, but its biological function in the intestine remains to be determined. Here we show that Olfm4 deletion induced colon adenocarcinoma in the distal colon of Apc<sup>Min/+</sup> mice. Mechanistically, we found that OLFM4 is a target gene of the Wnt/?-catenin pathway and can downregulate ?-catenin signaling by competing with Wnt ligands for binding to Frizzled receptors, as well as by inhibition of the Akt-GSK-3? (Akt-glycogen synthase kinase-3?) pathway. We have shown that both Wnt and nuclear factor-?B (NF-?B) signaling were boosted in tumor tissues of Apc Olfm4 double-mutant mice. These data establish OLFM4 as a critical negative regulator of the Wnt/?-catenin and NF-?B pathways that inhibits colon-cancer development initiated by APC mutation. In addition, Olfm4 deletion significantly enhanced intestinal-crypt proliferation and inflammation induced by azoxymethane/dextran sodium sulfate. Thus, OLFM4 has an important role in the regulation of intestinal inflammation and tumorigenesis, and could be a potential therapeutic target for intestinal malignant tumors. Unlike the human colonic epithelium, the mouse colonic epithelium does not express OLFM4, but nevertheless, systemic OLFM4 deletion promotes colon tumorigenesis and that loss from mucosal neutrophils may have a role to play.