Micheline J. Moussalli

Targeted Genetic Disruption of Peroxisome Proliferator–Activated Receptor-δ and Colonic Tumorigenesis

Peroxisome proliferator-activated receptor-delta (PPAR-delta) is overexpressed in human colon cancer, but its contribution to colonic tumorigenesis is controversial. We generated a mouse model in which PPAR-delta was genetically disrupted in colonic epithelial cells by targeted deletion of exon 4. Elimination of colon-specific PPAR-delta expression was confirmed by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), immunoblotting, and activity assays. Mice with and without targeted PPAR-delta genetic disruption (10-11 mice per group) were tested for incidence of azoxymethane-induced colon tumors. The effects of targeted PPAR-delta deletion on vascular endothelial growth factor expression were determined by real-time RT-PCR. Targeted PPAR-delta genetic disruption inhibited colonic carcinogenesis: Mice with PPAR-delta((-/-)) colons developed 98.5% fewer tumors than wild-type mice (PPAR-delta((-/-)) vs wild-type, mean = 0.1 tumors per mouse vs 6.6 tumors per mouse, difference = 6.5 tumors per mouse, 95% confidence interval = 4.9 to 8.0 tumors per mouse, P < .001, two-sided test). Increased expression of vascular endothelial growth factor in colon tumors vs normal colon was suppressed by loss of PPAR-delta expression. These findings indicate that PPAR-delta has a crucial role in promoting colonic tumorigenesis.

Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion

Loss of terminal cell differentiation promotes tumorigenesis. 15-Lipoxygenase-1 (15-LOX-1) contributes to terminal cell differentiation in normal cells. The mechanistic significance of 15-LOX-1 expression loss in human cancers to terminal cell differentiation suppression is unknown. In a screen of 128 cancer cell lines representing more than 20 types of human cancer, we found that 15-LOX-1 mRNA expression levels were markedly lower than levels in terminally differentiated cells. Relative expression levels of 15-LOX-1 (relative to the level in terminally differentiated primary normal human–derived bronchial epithelial cells) were lower in 79% of the screened cancer cell lines than relative expression levels of p16 (INK4A), which promotes terminal cell differentiation and is considered one of the most commonly lost tumor suppressor genes in cancer cells. 15-LOX-1 was expressed during terminal differentiation in three-dimensional air–liquid interface cultures, and 15-LOX-1 expression and terminal differentiation occurred in immortalized nontransformed bronchial epithelial but not in lung cancer cell lines. 15-LOX-1 expression levels were lower in human tumors than in paired normal lung epithelia. Short hairpin RNA–mediated downregulation of 15-LOX-1 in Caco-2 cells blocked enterocyte-like differentiation, disrupted tight junction formation, and blocked E-cadherin and ZO-1 localization to the cell wall membrane. 15-LOX-1 episomal expression in Caco-2 and HT-29 colon cancer cells induced differentiation. Our findings indicate that 15-LOX-1 downregulation in cancer cells is an important mechanism for terminal cell differentiation dysregulation and support the potential therapeutic utility of 15-LOX-1 reexpression to inhibit tumorigenesis. Cancer Prev Res; 4(12); 1961–72. ©2011 AACR.

Effects of Gut-Targeted 15-LOX-1 Transgene Expression on Colonic Tumorigenesis in Mice

Expression of 15-lipoxygenase-1 (15-LOX-1) is decreased in many human cancers; however, the mechanistic significance of its decreased expression has been difficult to determine because its mouse homolog 12/15-LOX has opposing functions. We generated a mouse model in which expression of a human 15-LOX-1 transgene was targeted to the intestinal epithelium via the villin promoter. Targeted expression was confirmed by real-time reverse transcription-polymerase chain reaction and immunoblotting. When the 15-LOX-1 transgene was expressed in colonic epithelial cells of two independent mouse lines (B6 and FVB), azoxymethane-inducible colonic tumorigenesis was suppressed (mean number of tumors: wild type [WT] = 8.2, 15-LOX-1(+/-) = 4.91, 15-LOX-1(+/+) = 3.57; WT vs 15-LOX-1(+/-) two-sided P = .003, WT vs 15-LOX-1(+/+) two-sided P < .001; n = 10-14 mice per group). 15-LOX-1 transgene expression was always decreased in the tumors that did develop. In the presence of expression of the 15-LOX-1 transgene, expression of tumor necrosis factor alpha and its target inducible nitric oxide synthase were decreased and activation of nuclear factor-kappa B in colonic epithelial cells was inhibited.

15-Lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway

The IL‐6/signal transducer and activator of transcription 3 (STAT3) pathway is a critical signaling pathway for colitis‐associated colorectal cancer (CAC). Peroxisome proliferator‐activated receptor (PPAR)‐δ, a lipid nuclear receptor, up‐regulates IL‐6. 15‐Lipoxygenase‐1 (15‐LOX‐1), which is crucial to production of lipid signaling mediators to terminate inflammation, down‐regulates PPAR‐δ. 15‐LOX‐1 effects on IL‐6/STAT3 signaling and CAC tumorigenesis have not been determined. We report that intestinally targeted transgenic 15‐LOX‐1 expression in mice inhibited azoxymethane‐ and dextran sodium sulfate‐induced CAC, IL‐6 expression, STAT3 phosphorylation, and IL‐6/STAT3 downstream target (Notch3 and MUC1) expression. 15‐LOX‐1 down‐regulation was associated with IL‐6 up‐regulation in human colon cancer mucosa. Reexpression of 15‐LOX‐1 in human colon cancer cells suppressed IL‐6 mRNA expression, STAT3 phosphorylation, IL‐6 promoter activity, and PPAR‐δ mRNA and protein expression. PPAR‐δ overexpression in colonic epithelial cells promoted CAC tumorigenesis in mice and increased IL‐6 expression and STAT3 phosphorylation, whereas concomitant 15‐LOX‐1 expression in colonic epithelial cells (15‐LOX‐1‐PPAR‐δ‐Gut mice) suppressed these effects: the number of tumors per mouse (mean ± sem) was 4.22 ± 0.68 in wild‐type littermates, 6.67 ± 0.83 in PPAR‐δ‐Gut mice (P= 0.026), and 2.25 ± 0.25 in 15‐LOX‐1‐PPAR‐δ‐Gut mice (P = 0.0006). Identification of 15‐LOX‐1 suppression of PPAR‐δ to inhibit IL‐6/STAT3 signaling‐driven CAC tumorigenesis provides mechanistic insights that can be used to molecularly target CAC.—Mao, F., Xu, M., Zuo, X., Yu, J., Xu, W., Moussalli, M. J., Elias, E., Li, H. S., Watowich, S. S., Shureiqi, I. 15‐Lipoxygenase‐1 suppression of colitis‐associated colon cancer through inhibition of the IL‐6/STAT3 signaling pathway. FASEB J. 29, 2359‐2370 (2015). www.fasebj.org

Potentiation of Colon Cancer Susceptibility in Mice by Colonic Epithelial PPAR-δ/β Overexpression

BACKGROUND The nuclear receptor peroxisome proliferator-activated receptor-δ/β (PPAR-d) is upregulated in human colorectal cancers, but its role in colonic tumorigenesis remains controversial. METHODS We generated a novel mouse model of intestinally targeted PPAR-d overexpression to simulate PPAR-d upregulation in human colon carcinogenesis. Colon-specific PPAR-d overexpression was confirmed by real-time reverse transcription polymerase chain reaction, immunoblotting, and activity assays. Mice with and without targeted PPAR-d overexpression were tested for azoxymethane (AOM)-induced colonic tumorigenesis. Mouse whole-genome transcriptome microarray analyses were performed to identify PPAR-d target genes to promote tumorigenesis. We used linear models to test for PPAR-d overexpression trend effects on tumor multiplicity. All statistical tests were two-sided. RESULTS Targeted PPAR-d overexpression markedly increased colonic tumor incidence (from 0 of 10 wild-type [WT] littermate mice to 9 of 10 mice [P < .001] in 2 FVB/N background mouse lines [villin-PPAR-d-1 and villin-PPAR-d-2] at a 5-mg/kg AOM dose) and multiplicity (number of tumors per mouse per mg/kg dose of AOM increased from 0.47 [95% confidence interval [CI] = 0.22 to 0.72] for the WT littermates to 2.15 [95% CI = 1.90 to 2.40] [P < .001] for the villin-PPAR-d-1 mice and from 0.44 [95% CI = 0.09 to 0.79] for the WT littermates to 1.91 [95% CI = 1.57 to 2.25] [P < .001] for the villin-PPAR-d-2 mice). PPAR-d overexpression reversed resistance to AOM-induced colonic tumorigenesis in C57BL/6 mice. PPAR-d overexpression modulated expression of several novel PPAR-d target genes in normal-appearing colonic epithelial cells of mice with PPAR-d overexpression in a pattern that matched the changes in colonic tumors. CONCLUSIONS Our finding that PPAR-d upregulation profoundly enhances susceptibility to colonic tumorigenesis should impact the development of strategies of molecularly targeting PPAR-d in cancer and noncancerous diseases.

Pleotropic effects of PPARD accelerate colorectal tumor progression and invasion

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.

PPARD regulation in gastric progenitor cells drives gastric tumorigenesis in mice

Little is known about the cell origin of gastric cancer. Peroxisome proliferator-activated receptor-delta (PPARD) is a druggable ligand-activated nuclear receptor that impacts protumorigenic cellular events. However, PPARD’s role in tumorigenesis, especially gastric tumorigenesis, remains to be defined. We found that targeting PPARD overexpression in murine gastric progenitor cells (GPC), via a villin promoter, spontaneously induced gastric tumorigenesis that progressed to invasive adenocarcinoma. PPARD overexpression in GPC upregulated tumorigenic proinflammatory cytokine and CD44 expression, expanded GPC population in vivo, enhanced GPC self-renewal and proliferation in organoid cultures, and endowed these cells with tumorigenic properties. Our findings identify PPARD as a driver of gastric tumorigenesis via GPC transformation.

Abstract 3819: PPAR-delta promotes Wnt/B-catenin-driven colorectal tumorigenesis

Introduction Aberrant Wnt/B-catenin signaling activation due to mutations in the adenomatous poliposis coli gene, APC, is a critical event in colorectal cancer (CRC). However, to drive CRC tumorigenesis, aberrant Wnt/B-catenin activation requires additional enhancing regulatory mechanisms. The peroxisome proliferator-activated receptor delta gene (PPARD), which is upregulated in CRC, is a downstream target of aberrant Wnt/B-catenin activation in human colon cancer cells. However, PPARD has also been reported to inhibit intestinal tumorigenesis in mice with germline APCmin mutations. Thus, the mechanistic interaction between PPARD and Wnt/β-catenin remains poorly understood. PPARD is a druggable protein for which agonists and antagonists are being developed. Determining PPARD effects on Wnt/B-catenin activation would define the direction of its therapeutic targeting in cancer and other diseases. Methods We developed a novel mouse model that simulates PPARD overexpression in CRC by inducing PPARD overexpression in intestinal epithelial cells via a villin promoter (villin-PPARD mice). We bred villin-PPARD mice with mice that have Apc580 mutation in intestinal epithelial cells induced by CDX2-Cre recombinase expression (Apc580mu mice) to generate Apc580mu-PPARD-Gut mice. Apc580mu-PPARD-Gut mice and Apc580mu control mice were monitored for CRC tumorigenesis. We also assessed the effects of PPARD expression modulation on Wnt/B-catenin activation in human colon cancer cell lines. Results PPARD overexpression in colonic epithelial cells increased CRC tumorigenesis in mice with Apc580 mutation. At 10 weeks of age, 100% of Apc580mu-PPARD-Gut mice but only 60% of Apc580mu mice had tumors of any size, and the mean number of tumors per Apc580mu-PPARD-Gut mouse (1.6 ± 2.5) was significantly higher than that per Apc580mu mouse (0.6 ± 0.25; P = 0.02). Tumors >3.5 mm were present in all Apc580mu-PPARD-Gut mice but only 20% of Apc580mu mice, and the mean number of tumors >3.5 mm per Apc580mu-PPARD-Gut mouse (1.4 ± 2.5) was significantly higher than that per Apc580mu mouse (0.2 ± 0.2; p = 0.005). In mice, colonic epithelial PPARD overexpression increased activated B-catenin protein levels and Wnt/B-catenin target gene mRNA levels. In human colon cancer cells, PPARD increased the levels of activated β-catenin, its nuclear localization and transcriptional activity. Conclusion Our findings indicate that PPARD augments Wnt/B-catenin signaling to promote CRC tumorigenesis and thus support the targeted inhibition of PPARD to suppress CRC tumorigenesis. Citation Format: Xiangsheng Zuo, Rui Tian, Shen Gao, Micheline J. Moussalli, Weidong Chen Chen, Ling Wu, Imad Shureiqi. PPAR-delta promotes Wnt/B-catenin-driven colorectal tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3819.

Abstract 1959: 15-lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway

The IL-6/STAT3 pathway is a critical signaling pathway for colitis-associated colorectal cancer (CAC). PPAR-delta (PPAR-d), a lipid nuclear receptor, upregulates IL-6. 15-Lipoxygenase-1 (15-LOX-1) is crucial to produce lipid signaling mediators to terminate inflammation. 15-LOX-1 downregulates PPAR-d. 15-LOX-1 effects on IL-6/STAT3 signaling and CAC tumorigenesis are unknown. We here report that intestinally targeted transgenic 15-LOX-1 expression in mice (15-LOX-1-Gut mice) inhibited azoxymethane and dextran sodium sulfate-induced CAC, IL-6 expression, STAT3 phosphorylation, and IL-6/STAT3 downstream target expression (Notch and MUC1). 15-LOX-1 downregulation was associated with IL-6 upregulation in human colon cancer mucosa. Re-expression of 15-LOX-1 in human colon cancer cells suppressed IL-6 mRNA expression, STAT3 phosphorylation, IL-6 promoter activity, and PPAR-d mRNA and protein expression. PPAR-d overexpression in colonic epithelial cells (PPAR-d-Gut mice) promoted CAC tumorigenesis in mice and increased IL-6 expression and STAT3 phosphorylation, whereas concomitant 15-LOX-1 expression in colonic epithelial cells (15-LOX-1-PPAR-d-Gut mice) suppressed these effects [e.g., tumor number per mouse (mean ± SE) was 4.22 ± 0.68 in wild-type littermates, 6.67 ± 0.83 in PPAR-d-Gut mice (p = 0.026), and 2.25 ± 0.25 in 15-LOX-1-PPAR-d-Gut mice (p = 0.0006)]. Identification of 15-LOX-1 suppression of PPAR-d to inhibit IL-6/STAT3 signaling-driven CAC tumorigenesis provides mechanistic insights that can be utilized to molecularly target CAC. Citation Format: Xiangsheng Zuo, Fei Mao, Min Xu, Weiguo Xu, Rui Tian, Micheline J. Micheline J. Moussalli, Elias Elias, Haiyan S. Li, Stephanie S. Watowich, Imad Shureiqi. 15-lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1959. doi:10.1158/1538-7445.AM2015-1959

Abstract 122: 15-lipoxygenase-1 regulation of HIF-1a and angiogenesis

Expression of 15-lipoxygenase-1(15-LOX-1) is down-regulated in colon and other major human cancer cells. 15-LOX-1 re-expression in cancer cells suppresses colonic tumorigenesis. We recently reported that targeted transgenic 15-LOX-1 expression in murine intestine suppresses colonic tumorigenesis. Nevertheless, the role of 15-LOX-1 loss of expression in cancer epithelial cells in metastases continues to be debated. Hypoxia-inducible factor 1-alpha (HIF-1a) is a transcriptional master regulator that enhances cancer cell survival under hypoxia and the development of various metastatic mechanisms (e.g. upregulation of VEGF to promote angiogenesis). We therefore examined the effects of 15-LOX-1 expression restoration in colon cancer cells on cell survival, migration and invasion, angiogenesis, and HIF-1a expression and stability under hypoxia. We found that 15-LOX-1 re-expression in colon cancer cells inhibited colon cancer cell survival, migration and invasion, VEGF expression, HUVEC cell tubular formation, and HIF-1a protein stability under hypoxia. These findings demonstrate that the loss of 15-LOX-1 significantly contribute to the ability of colon cancer cells to acquire metastasis promoting abilities by increasing a HIF-1 protein stability to up-regulate VEGF production of cancer cells and subsequently angiogenesis. Thus, therapeutic targeting 15-LOX-1 ubiquitous loss in cancer cells has the potential to be used to suppress tumor metastases. Citation Format: Yuanqing Wu, Fei Mao, Xiangsheng Zuo, Micheline J. Moussalli, Elias Elias, Imad Shureiqi. 15-lipoxygenase-1 regulation of HIF-1a and angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 122. doi:10.1158/1538-7445.AM2014-122