Pei-Li Yao

Activation of Peroxisome Proliferator-Activated Receptor-β/δ (PPAR-β/δ) Inhibits Human Breast Cancer Cell Line Tumorigenicity

The effect of activation and overexpression of the nuclear receptor PPAR-β/δ in human MDA-MB-231 (estrogen receptor–negative; ER−) and MCF7 (estrogen-receptor-positive; ER+) breast cancer cell lines was examined. Target gene induction by ligand activation of PPAR-β/δ was increased by overexpression of PPAR-β/δ compared with controls. Overexpression of PPAR-β/δ caused a decrease in cell proliferation in MCF7 and MDA-MB-231 cells compared with controls, whereas ligand activation of PPAR-β/δ further inhibited proliferation of MCF7 but not MDA-MB-231 cells. Overexpression and/or ligand activation of PPAR-β/δ in MDA-MB-231 or MCF7 cells had no effect on experimental apoptosis. Decreased clonogenicity was observed in both MDA-MB-231 and MCF7 overexpressing PPAR-β/δ in response to ligand activation of PPAR-β/δ as compared with controls. Ectopic xenografts developed from MDA-MB-231 and MCF7 cells overexpressing PPAR-β/δ were significantly smaller, and ligand activation of PPAR-β/δ caused an even greater reduction in tumor volume as compared with controls. Interestingly, the decrease in MDA-MB-231 tumor size after overexpressing PPAR-β/δ and ligand activation of PPAR-β/δ correlated with increased necrosis. These data show that ligand activation and/or overexpression of PPAR-β/δ in two human breast cancer cell lines inhibits relative breast cancer tumorigenicity and provide further support for the development of ligands for PPAR-β/δ to specifically inhibit breast carcinogenesis. These new cell-based models will be invaluable tools for delineating the role of PPAR-β/δ in breast cancer and evaluating the effects of PPAR-β/δ agonists. Mol Cancer Ther; 13(4); 1008–17. ©2014 AACR.

Peroxisome Proliferator-activated Receptor-D (PPARD) Coordinates Mouse Spermatogenesis by Modulating Extracellular Signal-regulated Kinase (ERK)-dependent Signaling

Background: The role of PPARD was examined in the testes because this receptor can regulate cell differentiation and proliferation. Results: PPARD temporally represses ERK-dependent cell cycle signaling and increases tight junction proteins in Sertoli cells. Conclusion: PPARD is essential for maturation of Sertoli cells to prevent testicular degeneration. Significance: PPARD modulates Sertoli cell function and spermatogenesis. Ppard−/− mice exhibit smaller litter size compared with Ppard+/+ mice. To determine whether peroxisome proliferator-activated receptor-D (PPARD) could possibly influence this phenotype, the role of PPARD in testicular biology was examined. Atrophic testes and testicular degeneration were observed in Ppard−/− mice compared with Ppard+/+ mice, indicating that PPARD modulates spermatogenesis. Higher expression of p27 and decreased expression of proliferating cellular nuclear antigen in Sertoli cells were observed in Ppard+/+ mice as compared with Ppard−/− mice, and these were associated with decreased Sertoli cell number in Ppard+/+ mice. Cyclin D1 and cyclin D2 expression was lower in Ppard+/+ as compared with Ppard−/− mice. Ligand activation of PPARD inhibited proliferation of a mouse Sertoli cell line, TM4, and an inverse agonist of PPARD (DG172) rescued this effect. Temporal inhibition of extracellular signal-regulated kinase (ERK) activation by PPARD in the testis was observed in Ppard+/+ mice and was associated with decreased serum follicle-stimulating hormone and higher claudin-11 expression along the blood-testis barrier. PPARD-dependent ERK activation also altered expression of claudin-11, p27, cyclin D1, and cyclin D2 in TM4 cells, causing inhibition of cell proliferation, maturation, and formation of tight junctions in Sertoli cells, thus confirming a requirement for PPARD in accurate Sertoli cell function. Combined, these results reveal for the first time that PPARD regulates spermatogenesis by modulating the function of Sertoli cells during early testis development.

Peroxisome proliferator-activated receptor-β/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation†

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator‐activated receptor‐β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over‐expressed in three human neuroblastoma cell lines, NGP, SK‐N‐BE(2), and IMR‐32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid‐induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All‐trans‐RA (atRA) decreased expression of SRY (sex determining region Y)‐box 2 (SOX2), a stem cell regulator and marker of de‐differentiation, in NGP and SK‐N‐BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR‐32 cells carrying wild‐type p53. Over‐expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK‐N‐BE(2), or IMR‐32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK‐N‐BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR‐32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over‐expressing PPARβ/δ induces cell differentiation through p53‐ and SOX2‐dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA‐resistant neuroblastoma patients.

Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-β/δ- and retinoic acid receptor-dependent mechanisms

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) has important physiological functions in control of cell growth, lipid and glucose homeostasis, differentiation and inflammation. To investigate the role of PPARβ/δ in cancer, stable human testicular embryonal carcinoma cell lines were developed that constitutively express PPARβ/δ. Expression of PPARβ/δ caused enhanced activation of the receptor, and this significantly decreased proliferation, migration, invasion, anchorage-independent growth, and also reduced tumor mass and volume of ectopic xenografts derived from NT2/D1 cells compared to controls. The changes observed in xenografts were associated with decreased PPARβ/δ-dependent expression of proliferating cell nuclear antigen and octamer-binding transcription factor-3/4, suggesting suppressed tumor proliferation and induction of differentiation. Inhibition of migration and invasion was mediated by PPARβ/δ competing with formation of the retinoic acid receptor (RAR)/retinoid X receptor (RXR) complex, resulting in attenuation of RARα-dependent matrix metalloproteinase-2 expression and activity. These results demonstrate that PPARβ/δ mediates attenuation of human testicular embryonal carcinoma cell progression through a novel RAR-dependent mechanism and suggest that activation of PPARβ/δ inhibits RAR/RXR dimerization and represents a new therapeutic strategy.

Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid

Previous studies suggested that perfluorooctanoate (PFOA) could activate the estrogen receptor (ER). The present study examined the hypothesis that PFOA can activate ER using an in vivo uterotrophic assay in CD-1 mice and an in vitro reporter assay. Pre-pubertal female CD-1 mice fed an estrogen-free diet from postnatal day (PND)14 through weaning on PND18 were administered 0, 0.005, 0.01, 0.02, 0.05, 0.1, or 1mg/kg PFOA or 17β-estradiol (E2, 0.5mg/kg) from PND18-20. In contrast to E2, PFOA caused no changes in the relative uterine weight, the expression of ER target genes, or the morphology of the uterus/cervix and/or vagina on PND21. Treatment of a stable human cell line containing an ER-dependent luciferase reporter construct with a broad concentration range of PFOA caused no change in ER-dependent luciferase activity; whereas E2 caused a marked increase of ER-dependent luciferase activity. These data indicate that PFOA does not activate mouse or human ER.

Editor’s Highlight: PPARβ/δ and PPARγ Inhibit Melanoma Tumorigenicity by Modulating Inflammation and Apoptosis

Skin tumorigenesis results from DNA damage, increased inflammation, and evasion of apoptosis. The peroxisome proliferator-activated receptors (PPARs) can modulate these mechanisms in non-melanoma skin cancer. However, limited data exists regarding the role of PPARs in melanoma. This study examined the effect of proliferator-activated receptor-β/δ (PPARβ/δ) and PPARγ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the UACC903 human melanoma cell line. Stable overexpression of either PPARβ/δ or PPARγ enhanced ligand-induced expression of a PPARβ/δ/PPARγ target gene in UACC903 cell lines as compared with controls. The induction of target gene expression by ligand activation of PPARγ was not altered by overexpression of PPARβ/δ, or vice versa. Stable overexpression of either PPARβ/δ or PPARγ reduced the percentage of cells in the G1 and S phase of the cell cycle, and increased the percentage of cells in the G2/M phase of the cell cycle in UACC903 cell lines as compared with controls. Ligand activation of PPARβ/δ did not further alter the distribution of cells within each phase of the cell cycle. By contrast, ligand activation of PPARγ enhanced these changes in stable UACC903 cells overexpressing PPARγ compared with controls. Stable overexpression of either PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/δ or PPARγ inhibited cell proliferation, and anchorage-dependent clonogenicity of UACC903 cell lines as compared with controls. Further, overexpression of either PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/δ or PPARγ inhibited ectopic xenograft tumorigenicity derived from UACC903 melanoma cells as compared with controls, and this was likely due in part to induction of apoptosis. Results from these studies demonstrate the antitumorigenic effects of both PPARβ/δ and PPARγ and suggest that targeting these receptors may be useful for primary or secondary melanoma chemoprevention.

Regulation of Cytochrome P450 2B10 (CYP2B10) Expression in Liver by Peroxisome Proliferator-Activated Receptor-β/δ Modulation of SP1 Promoter Occupancy

Alcoholic liver disease is a pathological condition caused by overconsumption of alcohol. Because of the high morbidity and mortality associated with this disease, there remains a need to elucidate the molecular mechanisms underlying its etiology and to develop new treatments. Because peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) modulates ethanol-induced hepatic effects, the present study examined alterations in gene expression that may contribute to this disease. Chronic ethanol treatment causes increased hepatic CYP2B10 expression inPparβ/δ+/+ mice but not in Pparβ/δ−/− mice. Nuclear and cytosolic localization of the constitutive androstane receptor (CAR), a transcription factor known to regulate Cyp2b10 expression, was not different between genotypes. PPARγ co-activator 1α, a co-activator of both CAR and PPARβ/δ, was up-regulated in Pparβ/δ+/+ liver following ethanol exposure, but not in Pparβ/δ−/− liver. Functional mapping of the Cyp2b10 promoter and ChIP assays revealed that PPARβ/δ-dependent modulation of SP1 promoter occupancy up-regulated Cyp2b10 expression in response to ethanol. These results suggest that PPARβ/δ regulates Cyp2b10 expression indirectly by modulating SP1 and PPARγ co-activator 1α expression and/or activity independent of CAR activity. Ligand activation of PPARβ/δ attenuates ethanol-induced Cyp2b10 expression in Pparβ/δ+/+ liver but not in Pparβ/δ−/− liver. Strikingly, Cyp2b10 suppression by ligand activation of PPARβ/δ following ethanol treatment occurred in hepatocytes and was mediated by paracrine signaling from Kupffer cells. Combined, results from the present study demonstrate a novel regulatory role of PPARβ/δ in modulating CYP2B10 that may contribute to the etiology of alcoholic liver disease.

Targeting estrogen receptor-β for the prevention of nonmelanoma skin cancer.

The potential for targeting estrogen receptor (ER)-β in various cancer models has been gaining considerable attention in recent years. In this issue of the journal, Chaudhary and colleagues demonstrate markedly decreased ultraviolet B (UVB)-induced skin cancer in a mouse model using a highly specific ER-β agonist, ERB-041. The mechanisms that underlie this strong inhibitory effect are mediated by inhibition of proinflammatory signaling and epithelial–mesenchymal transition (EMT). The changes in EMT were due in part to modulation of WNT/β-catenin signaling. Collectively, the results from these studies provide important new insights into the mechanisms by which the ER-β agonist ERB-041 inhibits UVB-induced skin cancer and opens the door for future studies that could examine combinatorial approaches for UVB-dependent skin cancer chemoprevention. Cancer Prev Res; 7(2); 182–5. ©2014 AACR.

Peroxisome proliferator-activated receptor-β/δ modulates mast cell phenotype.

The peroxisome proliferator‐activated receptor‐β/δ (PPARβ/δ) is known to have multiple anti‐inflammatory effects, typically observed in endothelial cells, macrophages, T cells and B cells. Despite the fact that mast cells are important mediators of inflammation, to date, the role of PPARβ/δ in mast cells has not been examined. Hence, the present study examined the hypothesis that PPARβ/δ modulates mast cell phenotype. Bone‐marrow‐derived mast cells (BMMCs) and peritoneal mast cells from Pparβ/δ+/+ mice expressed higher levels of high‐affinity IgE receptor (FcεRI) compared with Pparβ/δ−/− mice. BMMCs from Pparβ/δ+/+ mice also exhibited dense granules, associated with higher expression of enzymes and proteases compared with Pparβ/δ−/− mice. Resting BMMCs from Pparβ/δ+/+ mice secreted lower levels of inflammatory cytokines, associated with the altered activation of phospholipase Cγ1 and extracellular signal‐regulated kinases compared with Pparβ/δ−/− mice. Moreover, the production of cytokines by mast cells induced by various stimuli was highly dependent on PPARβ/δ expression. This study demonstrates that PPARβ/δ is an important regulator of mast cell phenotype.

Abstract 1295: Inhibition of clonogenicity and xenograft tumor growth by activation and/or over-expression of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ).

The peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) regulates a variety of biological processes but the function of PPARβ/δ in carcinogenesis remains controversial. Stable human cancer cell lines (A431, MDA-MB-231 and MCF7) that constitutively over-express PPARβ/δ were produced to effectively examine the role of this receptor in human cancer models. Ligand activation of PPARβ/δ both in MDA-MB-231 (ER-negative) and MCF7 (ER-positive) cells over-expressing PPARβ/δ cells caused significantly decreased clonogenicity compared to controls. Over-expression of PPARβ/δ reduced tumor volumes and tumor weights of ectopic xenografts from both MDA-MB-231 and MCF7cells compared to controls. Ligand activation of PPARβ/δ caused an even greater reduction of tumor volumes and tumor weights compared to controls. Interestingly, similar results were observed in the human epidermoid carcinoma cell line A431 and other human cancer cell lines. Ligand activation of PPARβ/δ strongly inhibited clonogenicity in A431 cells over-expressing PPARβ/δ. Xenografts from A431 control cells rapidly developed tumors while tumors were barely detectable in A431 cells over-expressing PPARβ/δ. These observations demonstrate the inhibitory effect of activating or over-expressing PPARβ/δ on tumorigenesis in human cancer cell lines. This suggests that PPARβ/δ suppression of tumorigenicity may be mediated through common mechanisms. Citation Format: Pei-Li Yao, Michael G. Borland, Prasad Krishnan, Bokai Zhu, Frank J. Gonzalez, Jeffrey M. Peters. Inhibition of clonogenicity and xenograft tumor growth by activation and/or over-expression of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1295. doi:10.1158/1538-7445.AM2013-1295