PAF and EZH2 induce Wnt/?-catenin signaling hyperactivation.
ABSTRACT: Fine control of Wnt signaling is essential for various cellular and developmental decision-making processes. However, deregulation of Wnt signaling leads to pathological consequences, one of which is cancer. Here, we identify a function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells and is required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing a secondary axis with ?-catenin target gene upregulation. Upon Wnt signaling activation, PAF dissociates from PCNA and binds directly to ?-catenin. Then, PAF recruits EZH2 to the ?-catenin transcriptional complex and specifically enhances Wnt target gene transactivation, independently of EZH2's methyltransferase activity. In mice, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling and propose a regulatory mechanism of Wnt signaling during tumorigenesis.
Project description:The underlying mechanisms of how self-renewing cells are controlled in regenerating tissues and cancer remain ambiguous. PCNA-associated factor (PAF) modulates DNA repair via PCNA. Also, PAF hyperactivates Wnt/?-catenin signaling independently of PCNA interaction. We found that PAF is expressed in intestinal stem and progenitor cells (ISCs and IPCs) and markedly upregulated during intestinal regeneration and tumorigenesis. Whereas PAF is dispensable for intestinal homeostasis, upon radiation injury, genetic ablation of PAF impairs intestinal regeneration along with the severe loss of ISCs and Myc expression. Mechanistically, PAF conditionally occupies and transactivates the c-Myc promoter, which induces the expansion of ISCs/IPCs during intestinal regeneration. In mouse models, PAF knockout inhibits Apc inactivation-driven intestinal tumorigenesis with reduced tumor cell stemness and suppressed Wnt/?-catenin signaling activity, supported by transcriptome profiling. Collectively, our results unveil that the PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively regulating self-renewing cells.
Project description:Deregulation of mitogen-activated protein kinase (MAPK) signaling leads to development of pancreatic cancer. Although Ras-mutation-driven pancreatic tumorigenesis is well understood, the underlying mechanism of Ras-independent MAPK hyperactivation remains elusive. Here, we have identified a distinct function of PCNA-associated factor (PAF) in modulating MAPK signaling. PAF is overexpressed in pancreatic cancer and required for pancreatic cancer cell proliferation. In mouse models, PAF expression induced pancreatic intraepithelial neoplasia with expression of pancreatic cancer stem cell markers. PAF-induced ductal epithelial cell hyperproliferation was accompanied by extracellular signal-regulated kinase (ERK) phosphorylation independently of Ras or Raf mutations. Intriguingly, PAF transcriptionally activated the expression of late endosomal/lysosomal adaptor, MAPK and mTOR activator 3 (LAMTOR3), which hyperphosphorylates MEK and ERK and is necessary for pancreatic cancer cell proliferation. Our results reveal an unsuspected mechanism of mitogenic signaling activation via LAMTOR3 and suggest that PAF-induced MAPK hyperactivation contributes to pancreatic tumorigenesis.
Project description:The enhancer of zeste homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/?-catenin signaling. We aimed at investigating EZH2's role in epigenetically regulating Wnt/?-catenin signaling following bacterial infection. NIH:Swiss outbred and Apc(Min/+) mice were infected with CR (10(8)?CFU); BLT1(-/-)Apc(Min/+) mice, azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated mice and de-identified human adenocarcinoma samples were the models of colon cancer. Following infection with wild-type but not mutant CR, elevated EZH2 levels in the crypt at days 6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and ?-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3's occupancy on WIF1 (Wnt inhibitory factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via small interfering RNA or EZH2-inhibitor deazaneplanocin A (Dznep) either alone or in combination with histone deacetylase inhibitor suberoylanilide hydroxamic acid, WIF1 promoter activity increased significantly while the overexpression of EZH2 attenuated WIF1 reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels, whereas H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, although ?-catenin levels were lower in EZH2-knocked down cells, F681Y mutants exhibited only partial reduction in ?-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days 6 and 12 post infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and ?-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected Apc(Min/+) mice paralleled changes recorded in BLT1(-/-)Apc(Min/+), AOM/DSS and human adenocarcinomas. Thus, EZH2-induced downregulation of WIF1 expression may partially regulate Wnt/?-catenin-dependent crypt hyperplasia in response to CR infection.
Project description:Wnt/?-catenin signaling is crucial for intestinal carcinogenesis and the maintenance of intestinal cancer stem cells. Here we identify the histone methyltransferase Mll1 as a regulator of Wnt-driven intestinal cancer. Mll1 is highly expressed in Lgr5<sup>+</sup> stem cells and human colon carcinomas with increased nuclear ?-catenin. High levels of MLL1 are associated with poor survival of colon cancer patients. The genetic ablation of Mll1 in mice prevents Wnt/?-catenin-driven adenoma formation from Lgr5<sup>+</sup> intestinal stem cells. Ablation of Mll1 decreases the self-renewal of human colon cancer spheres and halts tumor growth of xenografts. Mll1 controls the expression of stem cell genes including the Wnt/?-catenin target gene Lgr5. Upon the loss of Mll1, histone methylation at the stem cell promoters switches from activating H3K4 tri-methylation to repressive H3K27 tri-methylation, indicating that Mll1 sustains stem cell gene expression by antagonizing gene silencing through polycomb repressive complex 2 (PRC2)-mediated H3K27 tri-methylation. Transcriptome profiling of Wnt-mutated intestinal tumor-initiating cells reveals that Mll1 regulates Gata4/6 transcription factors, known to sustain cancer stemness and to control goblet cell differentiation. Our results demonstrate that Mll1 is an essential epigenetic regulator of Wnt/?-catenin-induced intestinal tumorigenesis and cancer stemness.
Project description:Estrogen receptors (ERs) [ERalpha (Esr1) and ERbeta (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERalpha and ERbeta is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERalpha knockout and Apc(Min) mouse strains, we demonstrate that ERalpha deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc(Min/+) mice. Within the normal intestinal epithelium of Apc(Min/+) mice, ERalpha deficiency is associated with an accumulation of nuclear beta-catenin, an indicator of activation of the Wnt-beta-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERalpha deficiency is associated with activation of Wnt-beta-catenin signaling, ERalpha deficiency in the intestinal epithelium of Apc(Min/+) mice also correlated with increased expression of Wnt-beta-catenin target genes. Through crosses between an ERbeta knockout and Apc(Min) mouse strains, we observed some evidence that ERbeta deficiency is associated with an increased incidence of colon tumors in Apc(Min/+) mice. This effect of ERbeta deficiency does not involve modulation of Wnt-beta-catenin signaling. Our studies suggest that ERalpha and ERbeta signaling modulate colorectal carcinogenesis, and ERalpha does so, at least in part, by regulating the activity of the Wnt-beta-catenin pathway.
Project description:The Wnt/?-catenin pathway plays a crucial role in development and renewal of the intestinal epithelium. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme in the synthesis of ketone body ?-hydroxybutyrate (?HB), contributes to the regulation of intestinal cell differentiation. Here, we have shown that HMGCS2 is a novel target of Wnt/?-catenin/PPAR? signaling in intestinal epithelial cancer cell lines and normal intestinal organoids. Inhibition of the Wnt/?-catenin pathway resulted in increased protein and mRNA expression of HMGCS2 and ?HB production in human colon cancer cell lines LS174T and Caco2. In addition, Wnt inhibition increased expression of PPAR? and its target genes, FABP2 and PLIN2, in these cells. Conversely, activation of Wnt/?-catenin signaling decreased protein and mRNA levels of HMGCS2, ?HB production, and expression of PPAR? and its target genes in LS174T and Caco2 cells and mouse intestinal organoids. Moreover, inhibition of PPAR? reduced HMGCS2 expression and ?HB production, while activation of PPAR? increased HMGCS2 expression and ?HB synthesis. Furthermore, PPAR? bound the promoter of HMGCS2 and this binding was enhanced by ?-catenin knockdown. Finally, we showed that HMGCS2 inhibited, while Wnt/?-catenin stimulated, glycolysis, which contributed to regulation of intestinal cell differentiation. Our results identified HMGCS2 as a downstream target of Wnt/?-catenin/PPAR? signaling in intestinal epithelial cells. Moreover, our findings suggest that Wnt/?-catenin/PPAR? signaling regulates intestinal cell differentiation, at least in part, through regulation of ketogenesis.
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.
Project description:B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear ?-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/?-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.
Project description:Aberrantly activated Wnt/β-catenin signaling pathway, as well as platelet-activating factor (PAF), contribute to cancer progression and metastasis of many cancer entities. Nonetheless, the role of the degradation enzyme named platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in ovarian cancer etiology is still unclear. This study investigated the functional impact of platelet-activating factor acetylhydrolase on BRCA1 mutant ovarian cancer biology and its crosstalk with the Wnt signaling pathway. PAF-AH, pGSK3β, and β-catenin expressions were analyzed in 156 ovarian cancer specimens by immunohistochemistry. PAF-AH expression was investigated in ovarian cancer tissue, serum of BRCA1-mutated patients, and in vitro in four ovarian cancer cell lines. Functional assays were performed after PLA2G7 silencing. The association of PAF-AH and β-catenin was examined by immunocytochemistry. In an established ovarian carcinoma collective, we identified PAF-AH as an independent positive prognostic factor for overall survival (median 59.9 vs. 27.4 months; <i>p</i> = 0.016). PAF-AH correlated strongly with the Wnt signaling proteins pGSK3β (Y216; nuclear: cc = 0.494, <i>p</i> < 0.001; cytoplasmic: cc = 0.488, <i>p</i> < 0.001) and β-catenin (nuclear: cc = 0.267, <i>p</i> = 0.001; cytoplasmic: cc = 0.291, <i>p</i> < 0.001). In particular, high levels of PAF-AH were found in tumor tissue and in the serum of BRCA1 mutation carriers. By in vitro expression analysis, a relevant gene and protein expression of PLA2G7/PAF-AH was detected exclusively in the BRCA1-negative ovarian cancer cell line UWB1.289 (<i>p</i> < 0.05). Functional assays showed enhanced viability, proliferation, and motility of UWB1.289 cells when PLA2G7/PAF-AH was downregulated, which underlines its protective character. Interestingly, by siRNA knockdown of PLA2G7/PAF-AH, the immunocytochemistry staining pattern of β-catenin changed from a predominantly membranous expression to a nuclear one, suggesting a negative regulatory role of PAF-AH on the Wnt/β-catenin pathway. Our data provide evidence that PAF-AH is a positive prognostic factor with functional impact, which seems particularly relevant in BRCA1 mutant ovarian cancer. For the first time, we show that its protective character may be mediated by a negative regulation of the Wnt/β-catenin pathway. Further studies need to specify this effect. Potential use of PAF-AH as a biomarker for predicting the disease risk of BRCA1 mutation carriers and for the prognosis of patients with BRCA1-negative ovarian cancer should be explored.