Progress 07/01/11 to 06/30/16
Outputs
Target Audience:Jeff Peters: New strategies to prevent and treat cancer continue to be a major priority. One of the goals of this project is to determine whether peroxisome proliferator-activated receptors (PPARs) can be used for cancer chemoprevention and/or chemotherapy. We identified natural compounds found in food sources, fatty acids, which may be suitable for this purpose. This requires comparison of synthetic compounds that are known to target PPARs before we can proceed with studies directed at natural products. For the past years of this project, we have made solid progress towards our goals that will impact many humans. Gary Perdew: The microbiome has recently been shown to play a critical role in the overall health and well being of humans. In addition, dysregulation of the microbiome contributes to the development of many chronic diseases. We have shown that the Ah receptor plays a key role in maintaining the microbiome, likely through local immune function in the gut. In the past year we have demonstrated for the first time that the lack of Ah receptor expression in a mouse model has a profound effect on the gut microbiome. Andrew Patterson: The gut microbiota modulates obesity and associated metabolic phenotypes in part through intestinal farnesoid X receptor (FXR) signaling. We developed glycine-β-muricholic acid (Gly-MCA), an intestinal FXR antagonist, to prevent or reverse high-fat diet (HFD)-induced and genetic obesity, insulin resistance, and fatty liver; however, the mechanism by which these phenotypes are improved is not understood. We report that modulation of the gut microbiota by inhibition of intestinal FXR signaling alters host liver lipid metabolism and improves obesity-related metabolic dysfunction. Curtis Omiecinski: Humans in industrialized areas are continuously exposed to phthalate plasticizers, prompting concerns of their potential toxicities. We assessed the relative activation potential of major diphthalate and their monophthalate metabolites against the human constitutive androstane receptor (hCAR) and its splice variants. We conclude that both di- and mono-phthalates are potent and selective hCAR2 activators and effective human pregnane X receptor (hPXR) activators, implicating these targets as important mediators of selective phthalate effects in humans. Adam Glick: Basic mechanisms of cancer development in epithelial tissues such as the skin continue to be an important priority, as these can lead to new therapeutic targets. We have identified the endoplasmic reticulum (ER) stress pathway initiated by the ER transmembrane kinase/RNase IRE1a as a critical to the response of normal skin cells to oncogenic stimuli. We have shown that this pathway regulates both the proliferative as well as senescence response to oncogene activation, and suggest that targeted modulation of this pathway could be important as an anti-cancer strategy. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctoral fellows, graduate students and undergraduate students were trained with these projects. How have the results been disseminated to communities of interest?Yes, the publications are freely available to all of the public on the internet. What do you plan to do during the next reporting period to accomplish the goals?Continue to examine the role of peroxisome proliferator-activated receptors, the constitutive androstane receptor, and the Ah receptor in carcinogenesis and/or homeostasis and how the gut microbiotia interacts with these receptors. Expand studies of ER stress in Ras driven cancers to in vivo models with genetically altered mice on the replacement project 4607.
Impacts
What was accomplished under these goals?
Jeff Peters: Results from these studies suggest that ligand activation of PPARbeta/delta attenuates hepatic tumorigenesis in HBV transgenic mice by inhibiting steatosis and cell proliferation, enhancing hepatocyte apoptosis, and modulating anti-inflammatory activity in Kupffer cells. Gary Perdew: Our results highlight a dominant regulatory role for the AHR in maintaining intestinal microbiota homeostasis and suggest that environmental modulation of AHR activity is likely to exert microbe-dependent effects upon host physiology. Andrew Patterson: Our results continue to demonstrate that the nuclear receptors like FXR are critical regulators of the signaling network existing between the host and the gut microbiota. Curtis Omiecinski: Our results demonstrate that hCAR2 is an important and potent target receptor for the prominent diphthalate plasticizers as well as their principle monophthalate metabolites, likely mediating their biological activities in humans. Adam Glick: Our results show that activation of the IRE1a ER stress pathway is critical to both oncogene induced cell proliferation and senescence. Modulating this pathway has the potential to be an important anticancer therapeutic strategy.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Balandaram, G., Kramer, L.R., Kang, B.-H., Murray, I.A., Perdew, G.H., Gonzalez, F.J. and Peters, J.M.. Ligand activation of peroxisome proliferator-activated receptor-?/? (PPAR?/?) attenuates hepatocarcinogenesis in hepatitis B transgenic mice. Toxicology (2016) 363: 1-9.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
I.A., Nichols, R.G., Zhang, L., Albert, I., Patterson, A.D., and Perdew, G.H. (2016) Expression of the aryl hydrocarbon receptor is necessary to maintain intestinal host-microbe homeostasis. Scientific Reports 6, 33969.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Zhang, L. Nichols, R.G., Chan, S.H.J, Jiang, C., Hao, R., Smith, P.B., Cai, J., Simons, M.N., Hatzakis, E., Maranas, C.D., Gonzalez, F.J., and Patterson, A.D. Farnesoid X Receptor Signaling Shapes the Gut Microbiota and Controls Hepatic Lipid Metbaolism. mSystems 2016 1(5): e00070-16.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Laurenzana, E.M., Coslo, D.M., Vigilar, M.V., Roman, A.M., and Omiecinski, C.J. Activation of the constitutive androstane receptor by monophthalates. Chemical Research in Toxicology (2016) 29:1651-1661.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Blazanin, N., Craig-Lucas, A., John, C., Breech, K., Son, J., Podolsky, M., and Glick, A. B. Endoplasmic reticulum stress and distinct outputs of the IRE1? RNase control proliferation and premature senescence in response to oncogenic Ras. Proc Nat Acad Sci USA.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Our target audience for this reporting period includes other investigators researching the role of peroxisome proliferator-activated receptors in carcinogenesis and homeostasis, exploring the role of the Ah receptor in inflammation and chronic diseases, assessing the regulation and mechanistic roles of CAR as a key mediator of toxicological response to xenobiotic exposures, and studying mechanisms of skin tumor induction and suppression. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctoral fellows, graduate students and undergraduate students were trained with these projects. How have the results been disseminated to communities of interest?The publications are freely available to all of the public on the internet. What do you plan to do during the next reporting period to accomplish the goals?Continue to examine the role of peroxisome proliferator-activated receptors, the constitutive androstane receptor, and the Ah receptor in carcinogenesis and/or homeostasis, and how gut microbiota influences interactions with these receptors.
Impacts
What was accomplished under these goals?
Results from these studies demonstrate that PPAR beta/delta attenuation of human testicular embryonal carcinoma cell progression is occurring through a novel RAR-dependent mechanism, suggesting that activation of PPAR beta/delta RAR/RXR dimerization represents a new therapeutic strategy for cancer. These findings provide new insights into the biochemical consequences of environmental contaminant exposure involving the alteration of the gut microbiota, modulation of nuclear receptor signaling, and disruption of host metabolism. Understanding these host:gut microbiome interactions provide important insight into how exposures through the diet can influence health and disease. The results examining the role of the Ah receptor in inflammatory signaling indicate that this receptor co-regulates, along with induction of NFkB, the expression of CCL20, a key mediator of T cell recruitment, to a site of infection. These results support the notion that the presence of bacterial ligands is sensed by the host leading to an enhanced response to a bacterial infection. These findings show the importance of the Ah receptor in normal epidermal differentiation. These studies open an additional avenue for the development of Ah receptor antagonists or selective modulators that can regulate Ah receptor signaling in keratinocytes to modulate epidermal differentiation and inflammation in a variety of skin inflammatory diseases. Results from our studies have identified gene activation profiles contributed by the constitutive androstane receptor (CAR) that underlie the mode of action of mouse liver carcinogenesis promoted by certain agricultural fungicide agents, such as propiconazole. Current research is directed at assessing differences in these responses between mice and humans.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Yao, P. L., Chen, L. P., Dobrzanski, T. P., Phillips, D. A., Zhu, B., Kang, B. H., Gonzalez, F. J. and Peters, J. M. Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-beta/delta- and retinoic acid receptor-dependent mechanisms. Oncotarget. 2015 Sep 26; doi: 10.18632/oncotarget.5415.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Zhang, L., Nichols, R. G., Correll, J., Murray, I. A., Tanaka, N., Smith, P. B., Hubbard, T. D., Sebastian, A., Albert, I., Hatzakis, E., Gonzalez, F. J., Perdew, G. H., and Patterson, A. D. Persistent organic pollutants modify gut microbiota-host metabolic homeostasis in mice through aryl hydrocarbon receptor activation. Environ Health Perspect. 2015 Jul;123(7):679-88. doi: 10.1289/ehp.1409055. Epub 2015 Mar 13. PubMed PMID: 25768209; PubMed Central PMCID: PMC4492271.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Lahoti, T. S., Boyer, J. A., Kusnadi, A., Muku, G. E., Murray, A. I., and Perdew, G. H. Aryl hydrocarbon receptor activation synergistically induces lipopolysaccharide-mediated expression of pro-inflammatory chemokine (c-c motif) ligand 20 (CCL20). Toxicol. Sci. 2015 Nov;148(1):229-40.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
van den Bogaard, E., Podolsky, M., Smits, J., Cui, X., John, C., Gowda, K., Desai, D., Amin, S., Schalkwijk, J., Perdew, G. H., and Glick, A. B. Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation. J Invest Dermatol. 2015 May;135(5):1320-8.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Currie, R. A., Peffer R. C., Goetz, A. K., Omiecinski, C. J., Goodman, J. I. Phenobarbital and propiconazole toxicogenomic profiles in mice show major similarities consistent with the key role that constitutive androstane receptor (CAR) activation plays in their mode of action. Toxicology 2014 Jul 3;321:80-88.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Our target audience for this reporting period includes other investigators researching the role of peroxisome proliferator-activated receptors in carcinogenesis and homeostasis, exploring the role of the Ah receptor in inflammation and chronic diseases, assessing the regulation and mechanistic roles of CAR as a key mediator of toxicological response to xenobiotic exposures, and studying mechanisms of skin tumor induction and suppression. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Postdoctoral fellows, graduate students and undergraduate students were trained under these projects. How have the results been disseminated to communities of interest? Manuscripts are freely available to the public (see products section), and graduate students and post-doctoral fellows presented their findings at meetings and conferences. What do you plan to do during the next reporting period to accomplish the goals? Continue to examine the role of peroxisome proliferator-activated receptors in carcinogenesis and homeostasis; to explore the role of the Ah receptor in inflammation and chronic diseases; to assess the regulation and mechanistic roles of CAR as a key mediator of toxicological response to xenobiotic exposures; and continue to study mechanisms of skin tumor induction and suppression.
Impacts
What was accomplished under these goals?
Endoplasmic reticulum stress and endoplasmic reticulum stress-associated unfolded protein response can promote cancer cell survival, but it remains unclear whether they can influence oncogene-induced senescence. This study examined the role of endoplasmic reticulum stress in senescence using oncogene-dependent models. Increased endoplasmic reticulum stress attenuated senescence in part by up-regulating phosphorylated protein kinase B and decreasing phosphorylated extracellular signal-regulated kinase. A positive feed-forward loop between phosphorylated protein kinase B, endoplasmic reticulum stress, and unfolded protein response was discovered, where a transient increase of endoplasmic reticulum stress caused reduced senescence and promotion of tumorigenesis. Further, decreased endoplasmic reticulum stress was correlated with increased senescence in both mouse and human tumors. Interestingly, H-RAS-expressing peroxisome proliferator-activated receptor-beta/delta null cells and tumors having increased cell proliferation exhibited enhanced endoplasmic reticulum stress, decreased cellular senescence, and/or enhanced tumorigenicity. Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Within the inflammatory milieu, resident fibroblast-like synoviocytes (FLS) in the synovial tissue undergo hyperplasia, which leads to joint destruction. Epidemiologic studies and our previous research suggest that activation of the aryl hydrocarbon receptor (AHR) pathway plays an instrumental role in the inflammatory and destructive RA phenotype. Thus, under inflammatory conditions, we hypothesized that the AHR is involved in the constitutive and inducible expression of several growth factors, FLS proliferation and migration, along with protease-dependent invasion in FLS from patients with RA (RA-FLS). Treatment with the AHR antagonist GNF351 inhibits cytokine-induced expression of vascular endothelial growth factor-A (VEGF-A), epiregulin, amphiregulin, and basic fibroblast growth factor mRNA through an AHR-dependent mechanism in both RA-FLS and FLS. Secretion of VEGF-A and epiregulin from RA-FLS also was inhibited upon GNF351 treatment. RA-FLS cell migration, along with cytokine-induced RA-FLS cell proliferation, was significantly attenuated by GNF351 exposure. Treatment of RA-FLS with GNF351 mitigated cytokine-mediated expression of matrix metalloproteinase-2 and -9 mRNA and diminished the RA-FLS invasive phenotype. These findings indicate that inhibition of AHR activity may be a viable therapeutic target in RA by attenuating growth factor release; FLS proliferation, migration, and invasion; and inflammatory activity. Functioning as a xenobiotic sensor, the constitutive androstane receptor (CAR; NR1I3) is predominantly expressed in the liver, and upon xenobiotic activation it modulates the expression of numerous hepatic genes that encode enzymes catalyzing phase I oxidation, phase II conjugation, and phase III drug transporter. Further, CAR also regulates pathways impinging upon the metabolism and secretion of endogenous molecules such as cholesterol and bilirubin, energy homeostasis, cell proliferation/apoptosis, and promotion of rodent hepatocarcinogenesis. Currently, the mechanisms that control CAR activation and nuclear translocation are poorly understood. Accumulating evidence suggests that the ubiquitin-proteasome system (UPS) functions in part to regulate nuclear receptor-mediated transcription. We hypothesized that the proteasome may similarly modulate CAR activation and its nuclear translocation. In this study, we demonstrate that proteasomal inhibition markedly disrupts CAR function, repressing CAR nuclear trafficking, disrupting CAR’s interaction with nuclear co-activators and inhibiting induction of CAR target gene responses in human primary hepatocytes following treatment with the xenobiotic CAR activators, phenobarbital and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Paradoxically, these effects occur following accumulation of ubiquitinated CAR and its interaction with the SUG1 subunit of the 26S proteasome. Together, our data show that the proteasome complex functions at multiple levels to regulate the functional biology of CAR activity, and we identified a novel role for the proteasomal complex as a critical mediator of CAR activation. In urine collected from 469 patients with lung cancer and 536 population controls, small molecules (<1,500 Da) were measured using unbiased liquid chromatography/mass spectrometry (LC/MS). Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points, and further confirmed in an independent sample set that comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared with the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA) were each significantly (P < 0.00001) elevated in non-small cell lung cancer and associated with worse prognosis [HR = 1.81 (P = 0.0002), and 1.54 (P = 0.025), respectively]. Creatine riboside was the strongest classifier of lung cancer status in all, and stage I-II cases, important for early detection, and also associated with worse prognosis in stage I-II lung cancer (HR = 1.71, P = 0.048). All measurements were highly reproducible, with intraclass correlation coefficients ranging from 0.82 to 0.99. Both metabolites were significantly (P < 0.03) enriched in tumor tissue compared with adjacent nontumor tissue (N = 48), thus revealing their direct association with tumor metabolism. Transforming growth factor beta 1 (TGFbeta1) is a pleiotropic cytokine in the skin that can function as a tumor promoter and suppressor in chemically-induced skin carcinogenesis, but the function in ultraviolet B (UVB) carcinogenesis is not understood well. Treatment of SKH1 hairless mice with the activin-like kinase 5 (ALK5) inhibitor SB431542 to block UVB-induced activation of cutaneous TGFbeta1 signaling suppressed skin tumor formation but did not alter tumor size or tumor cell proliferation. Tumors that arose in SB-treated mice after 30 weeks had significantly reduced percentage of IFNγ(+) tumor-infiltrating lymphocytes compared with control mice. SB431542 blocked acute and chronic UVB-induced skin inflammation and T-cell activation in the skin-draining lymph node (SDLN) and skin but did not alter UVB-induced epidermal proliferation. We tested the effect of SB431542 on migration of skin dendritic cell (DC) populations because DCs are critical mediators of T-cell activation and cutaneous inflammation. SB431542 blocked (i) UVB-induced Smad2 phosphorylation in dermal DC (dDC) and (ii) SDLN and ear explant migration of CD103(+) CD207(+) and CD207(-) skin DC subsets but did not affect basal or UV-induced migration of Langerhans cells. Mice expressing a dominant-negative TGFbeta type II receptor in CD11c(+) cells had reduced basal and UVB-induced SDLN migration of CD103(+) CD207(+) and CD207(-) DC subsets and a reduced percentage of CD86(high) dDC following UVB irradiation. Together, these suggest that TGFbeta1 signaling has a tumor-promoting role in UVB-induced skin carcinogenesis and this is mediated in part through its role in UVB-induced migration of dDC and cutaneous inflammation. Five journal articles on this research were published during this past year (see "products" section). And, a number of undergraduate and graduate students, and postdoctoral scholars, as well as other investigators were provided training under these research areas.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Zhu, B., Ferry, C.H., Markell, L.M., Blazanin, N., Glick, A.B., Gonzalez, F.J. and Peters, J.M. The Nuclear Receptor Peroxisome Proliferator-activated Receptor-beta/delta (PPAR beta/delta) Promotes Oncogene-induced Cellular Senescence through Repression of Endoplasmic Reticulum Stress. The Journal of Biological Chemistry (2014). 289: 20102-20119.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Lahoti, T.S., Hughes, J.M., Kusnadi, A., John, K., Zhu, B., Murray, I.A., Gowda, K., Peters, J.M., Amin, S., and Perdew, G.H. Ah receptor stimulates growth factor expression, proliferation and migration of fibroblast-like synoviocytes from rheumatoid arthritis patients. J. Pharmacol. Exp. Therapeutics (2014). 348:236-45.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Chen, T., Laurenzana, E.M., Coslo, D.M., Chen, F., Omiecinski, C.J. Proteosomal interaction as a critical activity modulator of the human constitutive androstane receptor. Biochemical Journal (2014). 458:95-107.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Mathe, E.A., Patterson, A.D., Haznadar, M., Manna, S.K., Krausz, K.W., Bowman, E.D., Shields, P.G., Idle, J.R., Smith, P.B., Anami, K., Kazandjian, D.G., Hatzakis, E., Gonzalez, F.J., and Harris, C.C. Non-invasive metabolomic profiling identifies diagnostic and prognostic markers in lung cancer. Cancer Research (2014). 74:3259-70.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Ravindran, A., Mohammed, J., Gunderson, A.J., Cui, X., and Glick, A.B. Tumor-promoting role of TGF?1 signaling in ultraviolet B-induced skin carcinogenesis is associated with cutaneous inflammation and lymph node migration of dermal dendritic cells. Carcinogenesis (2014). 35:959-960.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Individuals affected by cancers and environmental toxicant-induced damage may directly benefit as the findings may significantly change how these diseases are treated for prevention or therapy. Because peroxisome proliferator-activated receptors (PPARs) are activated by endogenous and dietary fatty acids, the findings may lead to future development of a variety of crops with specific fatty acids/lipids or the ability to enhance uptake of fatty acids/lipids that may be useful for chemoprevention and/or chemotherapy. AhR ligands are widespread in our diet and could enhance cancer outgrowth. Therefore, cancer researchers would be interested in the potential of an AhR antagonist to block tumor progression. Furthermore, they will benefit from the development of unique tools to understand the impact of diet and xenobiotic exposure are critical to fully elucidate how these and other receptors function under both normal and pathophysiologic conditions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Postdoctoral fellows and graduate students were trained in laboratory skills with this project. How have the results been disseminated to communities of interest? The manuscripts are freely available to the public. What do you plan to do during the next reporting period to accomplish the goals? Continue to examine the role of PPARs in toxicology, carcinogenesis and homeostasis. Examine the role of the Ah receptor in regulating inflammatory signaling in the presence of environmental contaminants. In addition, we will continue to elucidate the role of the constitutive androstane receptor (CAR) as a regulator of hepatoctye development and proliferation in relation to hepatocarcinogenesis.
Impacts
What was accomplished under these goals?
We examined the effect of prenatal perfluorooctanoic acid (PFOA) administration on pre- and postnatal development using peroxisome proliferator-activated receptor α (PPARα)-humanized mice to determine if there were species differences in receptor activity that might influence the developmental effects induced by PFOA. Pregnant mice were treated daily with water or PFOA (3 mg/kg) by oral gavage from gestation day 1 (GD1) until GD17 and then either euthanized on GD18, or allowed to give birth and then euthanized on postnatal day (PND) 20. No changes in average fetal weight, crown to rump length or placental weight were observed on GD18. Expression of mRNA encoding the PPARα target genes acyl CoA oxidase (Acox1) and cytochrome P450 4a10 (Cyp4a10) in maternal and fetal liver was increased on GD18 in wild-type and PPARα-humanized mice but not Pparα-null mice. On PND20, relative liver weight was higher in wild-type mice but not in Pparα-null mice or PPARα-humanized mice. Hepatic expression of Acox1 and Cyp4a10 mRNA was higher in wild-type mice but not in Pparα-null mice or PPARα-humanized mice on PND20. The percentage of mice surviving postnatally was lower in wild-type litters but not in litters from Pparα-null mice or PPARα-humanized mice. No changes in pup weight gain, onset of eye opening, or mammary gland development were found in any genotype. Previous studies in head and neck squamous cell carcinoma (HNSCC) cell lines have revealed the Ah receptor (AhR) to play a significant role in mediating the ‘aggressive’ phenotype of these cells. Therefore, this study sought to identify putative novel targets of the AHR associated with enhanced tumor invasiveness. Gene expression analysis identified three growth factor targets of AHR: amphiregulin (AREG), epiregulin (EREG) and platelet-derived growth factor A (PDGFA). Quantitative PCR analysis, ELISA and siRNA-mediated knock down of AhR revealed an attenuation of basal and/or induced levels of expression for these growth factors, in two HNSCC lines, following AhR antagonism. In silico analysis revealed these growth factors possess dioxin-like response elements. Two other AhR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene also elicited similar responses. In conclusion, this study identified AREG, EREG and PDGFA as growth factor targets of AhR activity associated with metastatic phenotype of HNSCC cells. These studies clearly demonstrate that AhR antagonism could be an important approach to treat cancer. Regulation of gene transcription is controlled in part by nuclear receptors that function coordinately through coregulator protein interactions. The human constitutive androstane receptor (CAR; NR1I3) is primarily expressed in the liver and regulates the expression of genes involved in xenobiotic metabolism as well as hormone, energy and lipid homeostasis. In our recent studies, we identified DAX-1, a nuclear receptor family member with corepressor properties, as a potent CAR regulator. Results of transaction and mutational studies demonstrated that both DAX-1’s downstream ‘LXXLL’ and its ‘PCFQVLP’ motifs were critical contributors to DAX-1’s co-repression activities. Recently, a role for DAX-1 as a regulator of liver physiology has emerged with its reported activity as a co-repressor of hepatocyte nuclear factor 4a and liver X receptor, resulting in negative regulation of gluconeogenic pathways and lipogenesis, respectively. Given the intersection of CAR with these pathways, the physiological impact of the DAX-1-CAR interactions is an important area of future investigation. The application of mass spectrometry-based metabolomics in the field of drug metabolism has yielded important insights into the metabolic routes of drugs and has provided unbiased, global perspectives of the endogenous metabolome that can be useful for identifying biomarkers associated with mechanism of action, efficacy, and toxicity. A stable isotope- and mass spectrometry-based metabolomics approach that captures both drug metabolism and changes in the endogenous metabolome in a single experiment is described in the publication. Here, the antioxidant drug tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) was chosen because its mechanism of action is not completely understood and its metabolic fate has not been studied extensively. Furthermore, its small size (MW = 172.2) and chemical composition (C(9)H(18)NO(2)) make it challenging to distinguish from endogenous metabolites. In this study, mice were dosed with tempol or deuterated tempol (C(9)D(17)HNO(2)) and their urine was profiled using ultraperformance liquid chromatography, coupled with quadrupole time-of-flight mass spectrometry. Principal component analysis of the urinary metabolomics data generated a Y-shaped scatter plot containing drug metabolites (protonated and deuterated) that were clearly distinct from the endogenous metabolites. Ten tempol drug metabolites, including eight novel metabolites, were identified. Phase II metabolism was the major metabolic pathway of tempol in vivo, including glucuronidation and glucosidation. Urinary endogenous metabolites significantly elevated by tempol treatment included 2,8-dihydroxyquinoline (8.0-fold, P < 0.05) and 2,8-dihydroxyquinoline-β-d-glucuronide (6.8-fold, P < 0.05). Urinary endogenous metabolites significantly attenuated by tempol treatment including pantothenic acid (1.3-fold, P < 0.05) and isobutrylcarnitine (5.3-fold, P < 0.01). This study underscores the power of a stable isotope- and mass spectrometry-based metabolomics in expanding the view of drug pharmacology. Numerous National Instititute of Health (NIH) grants have been secured by the investigators to help sustain research.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Albrecht, P.P., N.E. Torsell, P. Krishnan, D.J. Ehresman, S.R. Frame, S.-C. Chang, J.L. Butenhoff, G.L. Kennedy, F.J. Gonzalez and J.M. Peters. A species difference in the peroxisome proliferator-activated receptor alpha (PPARalpha)-dependent response to the developmental effects of perfluorooctanoic acid. Toxicological Sciences (2013) 131:568-582.
- Type:
Websites
Status:
Published
Year Published:
2013
Citation:
John, K., Lahoti, T.S., Wagner, K., Hughes, J.M., and G.H. Perdew. The Ah receptor regulates growth factor expression in head and neck squamous cell carcinoma cell lines. Mol. Carcinogenesis. Epub.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Li, F., Krausz, K.W., Jiang, C., Chen, C., Cook, J.A., Mitchell, J.B., Gonzalez, F.J., and A.D. Patterson. Stable isotope- and mass-spectrometry-based metabolomics as tools in drug metabolism: a study expanding tempol pharmacology. Journal of Proteome Research (2013) 12(3):1369-76.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Laurenzana, E.M., Chen, T., Kannuswamy, M., Sell, S.E., Strom, S.C., Li, Y., and C.J. Omiecinski. The orphan nuclear receptor, DAX-1, functions as a potent co-repressor of the constitutive androstane receptor (CAR; NR1I3). Molecular Pharmacology (2012) 82(5):918-928.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: The role of PPARbeta/delta in Harvey sarcoma ras (HRAS) expressing cells was examined. Ligand activation of PPARbeta/delta caused a negative selection against cells expressing higher levels of the HRAS oncogene by inducing a mitotic block. Mitosis-related genes that are predominantly regulated by E2F were induced to a higher level in HRAS-expressing PPARbeta/delta-null keratinocytes as compared to HRAS-expressing wild-type keratinocytes. Ligand activated PPARbeta/delta repressed expression of these genes by direct binding with p130/p107, facilitating nuclear translocation, and increasing promoter recruitment of p130/p107. We also coupled expression profiling with chromatin immunoprecipitation sequencing (ChIP-seq) to examine PPARbeta/delta-dependent regulation of gene expression in mouse keratinocytes, a cell type that expresses PPARbeta/delta in high concentration. Microarray analysis elucidated eight different types of regulation that modulated PPARbeta/delta-dependent gene expression of 612 genes ranging from repression or activation without an exogenous ligand, repression or activation with an exogenous ligand, or a combination of these effects. Bioinformatic analysis of ChIP-seq data demonstrated promoter occupancy of PPARbeta/delta for some of these genes, and also identified the presence of other transcription factor binding sites in close proximity to PPARbeta/delta bound to chromatin. For some types of regulation, ATF4 is required for ligand-dependent induction of PPARbeta/delta target genes.The aryl hydrocarbon receptor (AhR) has been shown to play a role in an increasing number of cellular processes. Recent reports have linked the AhR to cell proliferation, cytoskeletal arrangement, and tumor invasiveness in various tumor cell types. The AhR plays a role in the de-repression of the interleukin (IL) 6 promoter in certain tumor cell lines, allowing for increased transcriptional activation by cytokines. We have now demonstrated that there is a significant level of constitutive activation of the AhR in cells isolated from patients with head and neck squamous cell carcinoma (HNSCC). Constitutive activation of the AhR in HNSCCs was blocked by antagonist treatment, leading to a reduction in IL6 expression. In addition, the AhR exhibits a high level of expression in HNSCCs than in normal keratinocytes. These findings led to the hypothesis that the basal AhR activity in HNSCCs plays a role in the aggressive phenotype of these tumors and that antagonist treatment could mitigate this phenotype. This study provides evidence that antagonism of theAhR in HNSCC tumor cells, in the absence of exogenous receptor ligands, has a significant effect on tumor cell phenotype. Treatment of these cell lines with the AhR antagonists 6, 2, 4 -trimethoxyflavone, or the more potent GNF351, decreased migration and invasion of HNSCC cells and prevented benzo[a]pyrene-mediated induction of the chemotherapy efflux protein ABCG2. Thus, an AhR antagonist treatment has been shown to have therapeutic potential in HNSCCs through a reduction in aggressive cell phenotype. PARTICIPANTS: Jeffrey M. Peters, Dept. of Vet. & Biomed. Sci., Co-Principal Investigator, supervised the project and oversaw manuscripts. Gary Perdew, Dept. of Vet. & Biomed. Sci., Co-Principal Investigator, supervised the project and oversaw manuscripts. Adam Glick, Dept. of Vet. & Biomed. Sci., Associate Professor, supervised the project and writing manuscript. Combiz Khozoie, Dept. of Vet. & Biomed. Sci., Postdoc, research and writing manuscripts. Frank J. Gonzalez, National Cancer Institute, research and writing manuscripts. Moses Bility, Dept. of Vet. & Biomed. Sci., Graduate Student, research and writing manuscripts. Christina Ferry, Dept. of Biochem. Mol. BIol., Undergraduate Student, research and writing manuscripts. Bokai Zhu, Dept. of Vet. & Biomed. Sci., Graduate Student, research and writing manuscripts. Nicholas Blazanin, Dept. of Vet. & Biomed. Sci., Graduate Student, research and writing manuscripts, Brett C. DiNatale, Dept. of Vet. & Biomed. Sci., Graduate Student, research and writing manuscript. Kayla Smith, Dept. of Vet. & Biomed. Sci., Graduate Student, research and writing manuscript. Kaarthik John, Dept. of Vet. & Biomed. Sci., Post-doctoral fellow, research and writing manuscript. Gowdahalli Krishnegowda, Department of Pharmacology, Hershey Medical School, research associate, provided reagents. Shantu G. Amin, Department of Pharmacology, Hershey Medical School Professor, provided reagents. TARGET AUDIENCES: Individuals affected by cancers and environmental toxicant-induced damage may directly benefit as the findings may significantly change how these diseases are treated for prevention or therapy. Since PPARs are activated by endogenous and dietary fatty acids, the findings may lead to future development of a variety of crops with specific fatty acids/lipids or enhance uptake of fatty acids/lipids that may be useful for chemoprevention and/or chemotherapy. AHR ligands are widespread in our diet and could enhance cancer outgrowth and thus individuals would be interested in the potential of AHR antagonist to block tumor progression. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These results demonstrate a novel mechanism of PPARbeta/delta crosstalk with E2F signaling. Since co-treatment with a PPARbeta/delta ligand and various mitosis inhibitors increases the efficacy of increasing G2/M arrest, targeting PPARbeta/delta in conjunction with mitosis inhibitors could become a suitable option for development of new multi-target strategies for inhibiting RAS-dependent tumorigenesis. These results also demonstrate that PPARbeta/delta regulates constitutive expression of genes in keratinocytes, thus suggesting the presence of one or more endogenous ligands. The diversity in the types of gene regulation carried out by PPARbeta/delta is consistent with dynamic binding and interactions with chromatin and indicates the presence of complex regulatory networks in cells expressing high levels of this nuclear receptor such as keratinocytes. Results from these studies are the first to demonstrate that differences in DNA binding of other transcription factors can directly influence the transcriptional activity of PPARbeta/delta. The results in the Ah receptor (AHR) studies clearly indicate that antagonism of AHR activity in head and neck tumor cells dramatically decreases the aggressive phenotype of these cells. This firmly suggests that the AHR is a target for anti-metastatic therapy.
Publications
- Zhu, B., C. Khozoie, M. T. Bility, C. H. Ferry, N. Blazanin, A. B. Glick, F. J. Gonzalez, and J. M. Peters. 2012. Peroxisome proliferator-activated receptor-beta/delta cross talks with E2F and attenuates mitosis in HRAS-expressing cells. Molecular and Cellular Biology 32:2065-2082.
- DiNatale, B. C., K. Smith, K. John, G. Krishnegowda, S. G. Amin, and G. H. Perdew. 2012. Ah receptor antagonism represses head and neck tumor cell aggressive phenotype. Mol. Cancer Res. 10:1369-79.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) function and receptor cross-talk with other nuclear receptors, including PPARgamma and retinoic acid receptors (RARs), was examined using stable human HaCaT keratinocyte cell lines over-expressing PPARbeta/delta or PPARgamma. Enhanced ligand-induced expression of two known PPAR target genes, adipocyte differentiation-related protein (ADRP) and angiopoietin-like protein 4 (ANGPTL4), was found in HaCaT keratinocytes over-expressing PPARbeta/delta or PPARgamma. Over-expression of PPARbeta/delta did not modulate the effect of a PPARgamma agonist on up-regulation of ADRP or ANGPTL4 mRNA in HaCaT keratinocytes. All-trans retinoic acid (atRA) increased expression of a known RAR target gene, yet despite a high ratio of fatty acid binding protein 5 (FABP5) to cellular retinoic acid binding protein II, did not increase expression of ANGPTL4 or 3-phosphoinositide-dependent-protein kinase 1 (PDPK1), even in HaCaT keratinocytes expressing markedly higher levels of PPARbeta/delta. While PPARbeta/delta-dependent attenuation of staurosporine- or UVB-induced poly (ADP-ribose) polymerase (PARP) cleavage was not observed, PPARbeta/delta- and PPARgamma-dependent repression of UVB-induced expression and secretion of inflammatory cytokines was found in HaCaT keratinocytes over-expressing PPARbeta/delta or PPARgamma. There is increasing evidence that the aryl hydrocarbon receptor (AHR) plays a role in tumor progression through numerous mechanisms. We have previously shown that, in certain cancer cell lines that are typically nonresponsive to cytokine-mediated IL6 induction, activation of the AHR with the agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin derepresses the IL6 promoter and allows for synergistic induction following IL1 Beta treatment. The mechanism by which this occurs involves liganded AHR binding upstream from the transcription start site and dismissing HDAC-containing corepressor complexes, giving rise to a promoter structure that is more amenable to NF-κB activation. This fact, combined with observations of multiple endogenously produced chemicals activating the AHR, led us to study its role in basal expression among high cytokine-producing cancer cell lines. The current study provides evidence that several head and neck squamous cell carcinoma cell lines have a level of constitutively bound AHR at the IL6 promoter, allowing for higher basal and readily inducible IL6 transcription. Treatment of these cell lines with an AHR antagonist led to dismissal of the AHR from the IL6 promoter and recruitment of corepressor complexes, thus diminishing cytokine expression. Head and neck squamous cell carcinoma is typically a high cytokine-producing tumor type, with IL6 expression levels correlating with disease aggressiveness. For this reason, AHR antagonist treatment could represent a novel adjuvant therapy for patients, lowering pro-growth and antiapoptotic signaling with minimal systemic side effects. PARTICIPANTS: Jeffrey M. Peters, Dept. of Vet. & Biomed. Sci., Co-Principal Investigator, supervised the project and oversaw manuscripts. Gary Perdew, Dept. of Vet. & Biomed. Sci., Co-Principal Investigator, supervised the project and oversaw manuscripts. Combiz Khozoie, Dept. of Vet. & Biomed. Sci., Postdoc, research and writing manuscripts. Prajakta Albrecht, Dept. of Vet. & Biomed. Sci., Postdoc, research and writing manuscripts. Frank J. Gonzalez, National Cancer Institute, research and writing manuscripts. Michael Borland, Dept. of Vet. & Biomed. Sci., Graduate Student, research and writing manuscripts. Bokai Zhu, Dept. of Vet. & Biomed. Sci., Graduate Student, research and writing manuscripts. Tejas Lahoti, Dept. of Vet. & Biomed. Sci., Graduate Student, research and writing manuscripts. Christina Lee, Dept. of Vet. & Biomed. Sci., Undergrad, research and writing manuscripts. Iain Murray, Dept. of Vet. & Biomed. Sci., Senior Res. Assoc., research and writing manuscripts. Brett C. DiNatale, Dept. of Vet. & Biomed. Sci., Graduate Student, research and writing manuscripts. Jennifer C. Schroeder, Dept. of Vet. & Biomed. Sci., Postdoc, research and writing manuscripts. TARGET AUDIENCES: Individuals affected by cancers and environmental toxicant-induced damage may directly benefit as the findings may significantly change how these diseases are treated for prevention or therapy. Since PPARs are activated by endogenous and dietary fatty acids, the findings may lead to future development of a variety of crops with specific fatty acids/lipids or enhance uptake of fatty acids/lipids that may be useful for chemoprevention and/or chemotherapy. The AHR is modulated by the presence of AHR ligands in the gut both from the diet and produced by bacteria, this has major health implications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results from these studies suggest that FABP5 does not transport atRA or GW0742 to PPARbeta/delta and promote anti-apoptotic activity by increasing expression of PDPK1, or that PPARbeta/delta interferes with PPARgamma transcriptional activity. However, these studies demonstrate that stable over-expression of PPARbeta/delta or PPARgamma significantly increases the efficacy of ligand activation and represses UVB-induced expression of tumor necrosis factor alpha, interleukin 6, or interleukin 8 in HaCaT keratinocytes, thereby establishing an excellent model to study the functional role of these receptors in human keratinocytes.
Publications
- Borland, M. B., C. Khozoie, P. P. Albrecht, B. Zhu, C. Lee, T. S. Lahoti, F. J. Gonzalez, and J. M. Peters. 2011. Stable over-expression of PPARbeta/delta and PPARgamma to examine receptor signaling in human HaCaT keratinocytes. Cellular Signaling http://dx.doi.org/10.1016/j.cellsig.2011.07.020.
- DiNatale, B. C., J. C. Schroeder, and G. H. Perdew. 2011. Ah receptor antagonism inhibits constitutive IL6 production in head and neck tumor cell lines. Mol. Carcinog. 50(3):173-83. http://dx.doi.org/10.1002/mc.20702.
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