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APC mutations activate aberrant β-catenin signaling to drive initiation of colorectal cancer; however, colorectal cancer progression requires additional molecular mechanisms. PPAR-delta (PPARD), a downstream target of β-catenin, is upregulated in colorectal cancer. However, promotion of intestinal tumorigenesis following deletion of PPARD in Apcmin mice has raised questions about the effects of PPARD on aberrant β-catenin activation and colorectal cancer. In this study, we used mouse models of PPARD overexpression or deletion combined with APC mutation (ApcΔ580) in intestinal epithelial cells (IEC) to elucidate the contributions of PPARD in colorectal cancer. Overexpression or deletion of PPARD in IEC augmented or suppressed β-catenin activation via up- or downregulation of BMP7/TAK1 signaling and strongly promoted or suppressed colorectal cancer, respectively. Depletion of PPARD in human colorectal cancer organoid cells inhibited BMP7/β-catenin signaling and suppressed organoid self-renewal. Treatment with PPARD agonist GW501516 enhanced colorectal cancer tumorigenesis in ApcΔ580 mice, whereas treatment with PPARD antagonist GSK3787 suppressed tumorigenesis. PPARD expression was significantly higher in human colorectal cancer–invasive fronts versus their paired tumor centers and adenomas. Reverse-phase protein microarray and validation studies identified PPARD-mediated upregulation of other proinvasive pathways: connexin 43, PDGFRβ, AKT1, EIF4G1, and CDK1. Our data demonstrate that PPARD strongly potentiates multiple tumorigenic pathways to promote colorectal cancer progression and invasiveness.Significance:These findings address long-standing, important, and unresolved questions related to the potential role of PPARD in APC mutation-dependent colorectal tumorigenesis by showing PPARD activation enhances APC mutation-dependent tumorigenesis.

Supplementary Figures S1-S7 - This file includes supplementary figures S1-S7: Figure S1. PPARD increased active β-catenin and its target gene (e.g. c-Myc and cyclin D1) expression levels in mouse IECs and human HCT116 colorectal cancer cells by western blot and qRT-PCR. Figure S2. PPARD and its ligand GW501516 promoted intestinal tumorigenesis in multiple APC mutant mouse models, shown by intestinal photographs and tumor number counts. Figure S3. Western blot and IHCs showing that PPARD increased BMP7/TAK1/active β-catenin expression levels in mouse IECs. Figure S4. IHC microphotographs and score results showing active β-catenin expression levels in 41 human paired colorectal adenomas (Adenoma), CRC tumor centers (Tumor center), and cancer invasive fronts (Invasive front). Figure S5. Western blot and IHC results showing that PPARD upregulated AKT1 but not AKT2 to increase p-rpS6 levels in mouse IECs and human HCT116 colorectal cancer cells. Figure S6. Immunofluorescence staining of rRNA, IHC microphotographs and IHC score results of CDK1 showing rRNA expression levels in mouse intestinal tissues from PD and ApcÎ"580-PD mice and their corresponding control littermates, and nuclear CDK1 expression levels in 41 human paired colorectal adenomas (Adenoma), CRC tumor centers (Tumor center), and cancer invasive fronts (Invasive front). Figure S7. Multiple human public database analyses showing PPARD and BMP7 genetic alterations in colon cancer patients, and comparison of the survival probability for the colon cancer patients with low and high expression of BMP7.

APC mutations activate aberrant β-catenin signaling to drive initiation of colorectal cancer; however, colorectal cancer progression requires additional molecular mechanisms. PPAR-delta (PPARD), a downstream target of β-catenin, is upregulated in colorectal cancer. However, promotion of intestinal tumorigenesis following deletion of PPARD in Apcmin mice has raised questions about the effects of PPARD on aberrant β-catenin activation and colorectal cancer. In this study, we used mouse models of PPARD overexpression or deletion combined with APC mutation (ApcΔ580) in intestinal epithelial cells (IEC) to elucidate the contributions of PPARD in colorectal cancer. Overexpression or deletion of PPARD in IEC augmented or suppressed β-catenin activation via up- or downregulation of BMP7/TAK1 signaling and strongly promoted or suppressed colorectal cancer, respectively. Depletion of PPARD in human colorectal cancer organoid cells inhibited BMP7/β-catenin signaling and suppressed organoid self-renewal. Treatment with PPARD agonist GW501516 enhanced colorectal cancer tumorigenesis in ApcΔ580 mice, whereas treatment with PPARD antagonist GSK3787 suppressed tumorigenesis. PPARD expression was significantly higher in human colorectal cancer–invasive fronts versus their paired tumor centers and adenomas. Reverse-phase protein microarray and validation studies identified PPARD-mediated upregulation of other proinvasive pathways: connexin 43, PDGFRβ, AKT1, EIF4G1, and CDK1. Our data demonstrate that PPARD strongly potentiates multiple tumorigenic pathways to promote colorectal cancer progression and invasiveness. Significance: These findings address long-standing, important, and unresolved questions related to the potential role of PPARD in APC mutation-dependent colorectal tumorigenesis by showing PPARD activation enhances APC mutation-dependent tumorigenesis.

BACKGROUND & AIMS: The peroxisome proliferator activated receptor delta (PPARD) regulates cell metabolism, proliferation, and inflammation and has been associated with gastric and other cancers. Villin-positive epithelial cells are a small population of quiescent gastric progenitor cells. We expressed PPARD from a villin promoter to investigate the role of these cells and PPARD in development of gastric cancer. METHODS: We analyzed gastric tissues from mice that express the Ppard (PPARD1 and PPARD2 mice) from a villin promoter, and mice that did not carry this transgene (controls), by histology and immunohistochemistry. We performed cell lineage tracing experiments and analyzed the microbiomes, chemokine and cytokine production, and immune cells and transcriptomes of stomachs of these mice. We also performed immunohistochemical analysis of PPARD levels in in 2 sets of human gastric tissue microarrays. RESULTS: Thirty-eight percent of PPARD mice developed spontaneous, invasive gastric adenocarcinomas, with severe chronic inflammation. Levels of PPARD were increased in human gastric cancer tissues, compared with non-tumor tissues, and associated with gastric cancer stage and grade. We found an inverse correlation between level of PPARD in tumor tissue and patients survival time. Gastric microbiomes from PPARD and control mice did not differ significantly. Lineage-tracing experiments identified villin-expressing gastric progenitor cells (VGPCs) as the origin of gastric tumors in PPARD mice. In these mice, PPARD upregulated CCL20 and CXCL1, which increased infiltration of the gastric mucosa by immune cells. Immune cell production of inflammatory cytokines promoted chronic gastric inflammation and expansion and transformation of VGPCs, leading to tumorigenesis. We identified a positive-feedback loop between PPARD and interferon gamma signaling that sustained gastric inflammation to induce VGPC transformation and gastric carcinogenesis. CONCLUSIONS: We found PPARD overexpression in VPGCs to result in inflammation, dysplasia, and tumor formation. PPARD and VGPCs might be therapeutic targets for stomach cancer.

Colorectal carcinogenesis (CRC) progression requires additional molecular mechanisms to APC mutations/aberrant β-catenin signaling. PPARD is a druggable ligand-activated nuclear receptor that regulates essential genes involved in cell fate. PPARD is upregulated in intestinal epithelial cells (IECs) of human colorectal adenomas and adenocarcinomas. The mechanistic significance of PPARD upregulation in CRC remains unknown. Here we show that targeted PPARD overexpression in IECs of mice strongly augmented β-catenin activation via BMP7/TAK1 signaling, promoted intestinal tumorigenesis in Apcmin mice, and accelerated CRC progression and invasiveness in mice with IEC-targeted ApcΔ580 mutation. Human CRC invasive fronts had higher PPARD expression than their paired adenomas. A PPARD agonist (GW501516) enhanced APCΔ580 mutation-driven CRC, while a PPARD antagonist (GSK3787) suppressed it. Functional proteomics analyses and subsequent validation studies uncovered PPARD upregulation of multiple pro-invasive pathways that drive CRC progression (e.g. PDGFRβ, AKT1, CDK1 and EIF4G1). Our results identify novel mechanisms by which PPARD promotes CRC invasiveness and provide the rational for the development of PPARD antagonists to suppress CRC.

APC mutations activate aberrant β-catenin signaling to drive colorectal cancer (CRC) initiation. CRC progression however requires additional molecular mechanisms. PPAR-delta (PPARD) is a downstream target of β-catenin and upregulated in CRC. PPARD germline genetic deletion has however been reported to promote intestinal tumorigenesis in Apcmin mice, which has questioned PPARD effects on β-catenin aberrant activation and CRC. Addressing this knowledge gap is important because PPARD is a druggable protein. Mice with targeted PPARD overexpression/deletion into intestinal epithelial cells (IECs) were bred with mice with intestinally targeted ApcΔ580 mutation via CDX2-cre (ApcΔ580) or CDX2-cre-ERT2 (ApcΔ580-TMX). APCΔ580 mice treated with PPARD agonist (GW501516) or antagonist (GSK3787), and human CRC organoid cells were also tested. PPARD expression was examined in human CRC invasive fronts and their paired colorectal adenomas and cancer centers. PPARD’s mechanisms to promote CRC were screened by Functional Proteomics Reverse Phase Protein Assay (RPPA) and subsequently validated in in-vitro and in-vivo studies. PPARD overexpression/deletion in IECs in mice augmented/suppressed β-catenin activation via upregulation/downregulation of BMP7/TAK1 signaling and strongly promoted/suppressed CRC .PPARD downregulation by siRNA in human CRC organoid cells inhibited BMP7/β-catenin signaling and suppressed organoid self-renewals. GW501516 enhanced while GSK3787 suppressed CRC tumorigenesis in ApcΔ580 mice. PPARD expression was significantly higher in human CRC invasive fronts versus their paired invasive tumor centers and adenomas. RPPA and validation studies identified PPARD upregulation of multiple other pro-invasive pathways: PDGFRβ, connexin 43, AKT1, EIF4G1 and CDK1, Our data demonstrate that PPARD strongly potentiates multiple pro-tumorigenic pathways to promote CRC progression and invasiveness. Citation Format: Yi Liu, Yasunori Deguchi, Rui Tian, Daoyan Wei, Ling Wu, Weidong Chen, Weiguo Xu, Min Xu, Fuyao Liu, Shen Gao, Jonathan C. Jaoude, Sarah P. Chrieki, Micheline J. Moussalli, Mihai Gagea, Jeffrey S. Morris, Russell Broaddus, Xiangsheng Zuo, Imad Shureiqi. Pleiotropic effects of PPARD accelerate colorectal tumorigenesis progression and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2599.