Role of PPAR alpha in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator Wy-14,643

Chronic administration of peroxisome proliferators to mice and rats results in hepatomegaly and ultimately carcinogenesis. The mechanism underlying the carcinogenic effect of nongenotoxic peroxisome proliferators is not well understood. To determine whether nongenotoxic carcinogenesis is receptor mediated, we evaluated the effect of the prototypical peroxisome proliferator Wy-14,643 on replicative DNA synthesis and carcinogenesis in the PPAR alpha-null mouse line. Male mice (F4, Sv/129 ter) of both genotypes (+/+) and (-/-) were fed either a control diet or one containing 0.1% Wy-14,643 for either 1 week, 5 weeks, or 11 months. Wild-type mice fed the Wy-14,643 diet for 1 or 5 weeks showed increased hepatic labeling by bromodeoxyuridine (BrDU) compared to untreated controls. In contrast, there was no increase in hepatic BrDU labeling index in (-/-) mice fed the Wy-14,643 diet for the same time periods compared to controls. After 11 months, 100% of the (+/+) mice fed the Wy-14,643 diet had multiple hepatocellular neoplasms, including adenomas and carcinomas, while the (-/-) mice fed the Wy-14,643 diet were unaffected. This work demonstrates that the effects of Wy-14,643 on replicative DNA synthesis and hepatocarcinogenesis are mediated by PPAR alpha.      

TGF{alpha} is differentially expressed in liver foci induced by diethylnitrosamine initiation and peroxisome proliferator promotion

Transforming growth factor     

Oxidants from nicotinamide adenine dinucleotide phosphate oxidase are involved in triggering cell proliferation in the liver due to peroxisome proliferators

It was shown that 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (Wy-14,643), a potent peroxisome proliferator, caused rapid oxidant-dependent activation of nuclear factor kappaB (NF-kappaB) in Kupffer cells in vivo and activated superoxide production by isolated Kupffer cells. Here, we tested the hypothesis that NADPH oxidase (NADPH OX) is the source of oxidants increased by Wy-14,643. Indeed, both activation of NF-kappaB and increases in cell proliferation due to a single dose of Wy-14,643 (100 mg/kg) were prevented completely when rats were pretreated with diphenyleneiodonium (1 mg/kg), an inhibitor of NADPH OX. p47phox is a critical subunit of NADPH OX; therefore, p47phox knockout mice were used to specifically address the hypothesis of NADPH OX involvement. In livers of wild-type mice, Wy-14,643 activated NF-kappaB, followed by an increase in mRNA for tumor necrosis factor a. Importantly, these changes did not occur in p47phox knockouts. Moreover, when Kupffer cells were treated with Wy-14,643 in vitro, superoxide production was increased in cells from wild-type but not p47phox-null mice. Finally, when mice were fed a Wy-14,643-containing (0.1%) diet for 7 days, the increase in liver weight and cell proliferation caused by Wy-14,643 in wild-type mice was blocked in p47phox-null mice. Combined, these results are consistent with the hypothesis that Wy-14,643 activates NADPH OX, which leads to NF-kappaB-mediated production of mitogens that causes hepatocellular proliferation characteristic of this class of nongenotoxic carcinogens.     

Antibodies to tumor necrosis factor alpha prevent increases in cell replication in liver due to the potent peroxisome proliferator, WY- 14,643

Several structurally dissimilar hypolipidemic drugs, plasticizers and halogenated hydrocarbons induce peroxisomes in hepatocytes, and cause hepatocellular adenoma and carcinoma in rats and mice. The mechanism by which these agents act is unknown, although recent studies have suggested a link between increased cell proliferation and hepatic cancer caused by peroxisome proliferators. Here, we demonstrate that neutralizing antibodies to tumor necrosis factor alpha (TNF alpha) block increases in protein kinase C and cell proliferation due to [4- chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY-14,643), a hypolipidemic drug and potent peroxisome proliferator that causes tumors. WY-14,643 moderately elevated the level of TNF alpha mRNA in the liver. TNF alpha was detected immunohistochemically exclusively in Kupffer cells. These results demonstrate that WY-14,643 acts as an indirect mitogen on hepatocytes via TNF alpha. We propose that the Kupffer cell, a major source of TNF alpha in the liver, is involved in the mechanism of the mitogenic effect of WY-14,643.      

Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: relationship to mitochondrial alterations

Recent studies have indicated a lack of correlation betweenhepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and thecarcinogenicity of peroxisome proliferators (PP) and suggestedthat DNA in intact hepatic nuclei may be insensitive to increasesin 8-OHdG resulting from PP exposure. The possibility that PP-inducedelevations in acyl CoA oxidase (ACO) activity might result inoxidative damage to mitochondrial DNA (mtDNA) was thereforeinvestigated by feeding male F344 rats the hepatocarcinogenicPP Wy-14,643 (Wy, 0.1% in the diet) for 3, 6, 11, or 22 weeks,or clofibric acid (CA, 0.5% in the diet) for 22 weeks. Followingthe respective PP exposures, hepatic peroxisomal acyl CoA oxidaseactivity was determined and DNA isolated from either mitochondriaor unfractionated liver homogenates and analysed for the presenceof 8-OHdG. PP treatment caused an increase in ACO activity (10-to 15-fold) at all time points examined and an increase of 8-OHdG(1.5- to 2-fold) in DNA isolated from unfractionated liver homogenatesfollowing PP treatment for 11 or 22 weeks. No increase of 8-OHdGin mtDNA was detected. However, quantitation of a PCR amplifiedregion from the D-loop of mtDNA demonstrated a 2- to 3-foldincrease in the relative amount of mtDNA in DNA isolated fromunfractionated liver homogenates following 3, 11, and 22 weeksexposure to Wy or CA (22 weeks only). In addition, a slightincrease in the mitochondrial volume density (1.4-fold) wasobserved in electron micrographs of liver samples from ratsexposed to Wy for 22 weeks. These results (i) demonstrate thatPP treatment, at levels which cause an increase in ACO activity,does not cause oxidative damage to mtDNA, and (ii) suggest thatone reason for the observed increase of 8-OHdG in DNA from unfractionatedliver homogenates may be an increase in the amount of mtDNApresent in these samples. Furthermore, these studies provideadditional evidence against a role of oxidative DNA damage,measured as 8-OHdG, in PP-induced rodent hepatocarcinogenesisand suggest that alterations in mitochondria or other effectsmay be more pertinent to PP-related carcinogenesis.     

Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: relationship to carcinogenesis and nuclear localization

Increased oxidative DNA damage due to increased peroxisomalgeneration of H₂O₂ is a potential mechanism in the carcinogenicityof chemical peroxisome proliferators (PP) in rodent liver. Inorder to determine the relationship between carcinogencity andperoxisome-dependent DNA damage, levels of DNA base oxidationwere examined by comparing 8-hydroxydeoxyguanosine (8-OHdG)in DNA from unfractionated liver of male F344 rats followingdietary exposure to PP [WY-14, 643, 0.1% or 0.005%; di(2-ethylhexyl)phthalate(DEHP), 1.2%; clofibric acid, 0.5%] or phenobarbital (0.05%).Exposure-related increases in 8-OHdG were not observed at 3or 11 weeks for any of the compounds fed. At 22 weeks, 8-OHdGwas similarly elevated (2–3x) by WY-14, 643 (0.1% and0.005%) and clofibric acid (0.5%). These equivalent increasesin 8-OHdG in DNA from unfractionated liver did not parallelthe divergent carcinogencity of these different dietary exposuresin the present or previous studies. The potential oxidationof nuclear DNA was examined by comparing levels of 8-OHdG inDNA isolated from purified liver nuclei and unfractionated liver.Elevated levels of 8-OHdG were not detected in DNA isolatedfrom nuclear fractions of livers from rats fed clofibric acidfor 22 weeks, indicating the dependence of PP-induced oxidativeDNA damage on extranuclear components of samples for DNA isolation.The absence of a quantitative relationship between PP-inducedcarcinogenicity and oxidative DNA base damage (as 8-OHdG), andthe failure to localize this oxidative damage to nuclear DNA,suggest two possible conclusions: (1) quantitation of 8-OHdG,a specific and sensitive indicator of oxidative DNA damage,does not accurately reflect the potential peroxisomal H₂O₂-dependentDNA damage and carcinogenicity of PP exposure in rodents; (2)other hepatic responses may be more critical features of themechanism of PP carcinogenicity.     

Overexpression of the peroxisome proliferator activated receptor alpha or the human c-Ha-ras transgene is not involved in tumorigenesis induced by di(2-ethylhexyl)phthalate in rasH2 mice

The mRNA profiles for peroxisome proliferator activated receptor (PPAR) alpha and human c-Ha-ras genes were determined by real-time semi-quantitative polymerase chain reaction analysis of hepatocellular adenomas induced by di(2-ethylhexyl)phthalate (DEHP) in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice). The mRNA levels were essentially equal in hepatocellular adenomas and adjacent non-neoplastic hepatocytes, in spite of a remarkable elevation in the cell proliferation index in tumors determined by anti-Ki-67 immunohistochemistry. From the results, it is concluded that overexpression of PPARalpha or the transgene is not associated with the liver tumorigenesis induced by DEHP in rasH2 mice.     

Effect on the expression of c-met, c-myc and PPAR-{alpha} in liver and liver tumors from rats chronically exposed to the hepatocarcinogenic peroxisome proliferator WY-14, 643

The induction of rodent hepatic tumors by peroxisome proliferators(PP) appears to depend on focal growth of hepatocytes. Expressionof the oncogenes c-met and c-mycis altered following regenerativestimuli in rat liver, suggesting involvement of their proteinproducts in hepatocyte replication. In addition, increases inc-myc and c-met mRNA expression are observed in multiple typesof human and rodent tumors, including hepatocellular carcinoma.A study was designed to test the hypothesis that developmentof PP-induced hepatic neoplasms occurs as a result of overexpressionof c-met or c-myc. Male F344 rats were exposed to WY-14, 643for 22 or 78 weeks (1000 p.p.m. in the diet). Messenger RNAwas extracted from liver tumors (78 weeks) and surrounding non-lesionliver of exposed rats and non-lesion liver from age-matchedcontrol rats. Levels of mRNA expression were compared usingNorthern analysis. Significant increases in c-met (     

The A9 antigen associated with aggressive human squamous carcinoma is structurally and functionally similar to the newly defined integrin alpha 6 beta 4.

We previously reported that altered expression of the A9 antigen (defined by monoclonal antibody UM-A9) is a predictive marker of early recurrence and progression of squamous cell carcinoma (SCC). In normal squamous cells A9 expression is limited to the site of contact with the basement membrane in vivo and the culture surface in vitro, whereas aggressive SCCs exhibit loss of polarity and increased intensity of A9 expression. The potential relationship of the A9 antigen to structures known to be involved in cell adhesion was analyzed by immunobiochemical and cell adhesion assays. UM-A9 precipitates a complex of protein chains reminiscent of the alpha and beta heterodimer glycoproteins that characterize the integrin family of extracellular matrix receptors. Proteins were isolated from A9-positive cells using UM-A9 and well-defined antibodies specific for integrin alpha and beta chains. UM-A9, anti-alpha 6, and anti-beta 4 monoclonal antibodies (mAbs) all precipitated proteins with comparable electrophoretic mobilities. Furthermore, UM-A9 mAb precleared the SCC alpha 6 beta 4 integrin complex isolated with anti-alpha 6 or anti-beta 4 mAbs but not that isolated by anti-beta 1 mAb. The isoelectric points of the A9 complex chains were consistent with those reported for alpha 6 and beta 4. Three of the polypeptide chains (140, 175, and 205 kDa) precipitated by UM-A9 showed peptide homology to one another and to the beta 4 chain precipitated by mAb 439-9B. The A9/alpha 6 subunit is composed of 125- and 30-kDa chains and was distinguished from beta 4 and beta 1 chains by its peptide map and isoelectric point. UM-A9 binds to an epitope common to the beta 4 subunits since in pulse-chase analysis the beta 4 species are precipitated at an early time point, whereas detection of alpha-subunit synthesis is detected during assembly of the mature complex. Immunoprecipitation and preclearing experiments demonstrated that in SCC the alpha 6 subunit is associated primarily with the beta 4 species and not with the 130-kDa beta 1 subunit. In cell adhesion assays on extracellular matrix proteins, the alpha 6-specific GoH3 mAb inhibited binding of SCC to laminin, suggesting that alpha 6 beta 4 may function as a laminin receptor in SCC. These data and our prior observations showing an association between altered A9 expression and early recurrence in SCC provide the first evidence that altered expression of alpha 6 beta 4 integrin is associated with the clinical behavior of human squamous cell carcinomas.     

Expression of base excision DNA repair genes is a sensitive biomarker for in vivo detection of chemical-induced chronic oxidative stress: identification of the molecular source of radicals responsible for DNA damage by peroxisome proliferators

Oxidative stress to DNA is recognized as one of the mechanisms for the carcinogenic effects of some environmental agents. Numerous studies have been conducted in an attempt to document the fact that chemical carcinogens that are thought to induce production of oxidants also cause the formation of oxidative DNA lesions. Although many DNA adducts continue to be useful biomarkers of dose/effect, changes in gene expression have been proposed to be a practical novel tool for studying the role of chemically induced oxidative DNA damage. Here, we hypothesized that expression of base excision DNA repair genes is a sensitive biomarker for in vivo detection of chemically induced chronic oxidative stress. To test this hypothesis, mice were treated with a known rodent carcinogen and peroxisome proliferator, WY-14,643 (500 ppm, 1 month). A number of end points that are commonly used to assess oxidative DNA damage were considered. Our data demonstrate that no difference in 8-oxoguanine, the number of abasic sites, or single strand breaks can be detected in genomic DNA from livers of control or WY-treated animals. However, a concordant marked induction of genes specific for the long-patch base excision DNA repair, a predominant pathway that removes oxidized DNA lesions in vivo, was observed in livers of WY-treated mice. Kupffer cell NADPH oxidase, and peroxisomes in parenchymal cells have been proposed as the potential sources of peroxisome proliferator-induced oxidants. The analysis of expression of base excision DNA repair genes was used to assess whether this biomarker of oxidative stress can be used to determine the source of oxidants. The data suggest that DNA-damaging oxidants are generated by enzymes that are induced after activation of peroxisome proliferator activator receptor alpha, such as those involved in lipid metabolism in peroxisomes, and are not the result of activation of NADPH oxidase in Kupffer cells. We conclude that expression of base excision DNA repair genes is a sensitive in vivo biomarker for chemically induced oxidative stress to DNA that can be successfully used for the identification of the molecular source of radicals responsible for DNA damage in vivo.     

The truncated glucocorticoid receptor in the P1798 mouse lymphosarcoma is associated with resistance to glucocorticoid lysis but not to other glucocorticoid-induced functions.

Glucocorticoid receptors in lines of the P1798 mouse lymphosarcoma either sensitive or resistant to glucocorticoid-induced lysis have been characterized and their functional significance determined. The glucocorticoid receptor from the cortisol-sensitive tumor is an Mr approximately 98,000 protein with a Stokes radius of 7.4 nm in the oligomeric, non-DNA-binding state and 5.6 nm in the transformed, DNA-binding state. This receptor binds glucocorticoid and reacts with the BUGR-2 monoclonal antibody. In contrast, two abnormal receptor species were identified in the cortisol-resistant tumor. One is an Mr approximately 98,000 non-steroid-binding but immunologically reactive protein. The other is an Mr approximately 45,000 species which contains both steroid- and DNA-binding sites but exhibits little or no reactivity with BUGR-2, suggesting that its NH2 terminus is truncated in a region within or adjacent to the BUGR epitope. This species had Stokes radii of 5.8 and 3.5 nm in nontransformed and transformed states, respectively. In both tumor lines, glucocorticoids stimulated the activities of glutamine synthetase and 5'-nucleotidase and the synthesis of glucocortin. However, glucocorticoid-induced tumor regression occurred only in the cortisol-sensitive tumor. Additionally, the glucocorticoid inducibility of a specific protein in the sensitive, but not in the resistant, tumor was demonstrated, as well as the presence of a protein specific to the resistant line. Taken together, these results suggest that the truncated glucocorticoid receptor in the P1798 lymphosarcoma is functional, although possibly in a more restricted gene-specific manner, and that the lysis defect, while possibly resulting from a truncated receptor, may also result from the inability of glucocorticoids to induce a critical protein in the pathway of programmed cell death and/or from the presence of a protein which inhibits the lytic response.     

The PDGF pathway in breast cancer is linked to tumour aggressiveness,triple-negative subtype and early recurrence

Purpose

The platelet-derived growth factor (PDGF) signalling pathway is often dysregulated in cancer and PDGF-receptor expression has been linked to unfavourable prognostic factors in breast cancer (e.g. ER negativity, high Ki67 and high grade). This study aimed to evaluate the expression of PDGFRα, PDGFRβ and ligand PDGF-CC in breast cancer in relation to molecular subtypes and prognosis.

Methods

Protein expression of tumour and/or stromal cell PDGFRα, PDGFRβ and PDGF-CC was evaluated in primary tumours (N = 489), synchronous lymph node metastases (N = 135) and asynchronous recurrences (N = 39) using immunohistochemistry in a prospectively maintained cohort of primary breast cancer patients included during 1999–2003. Distant recurrence-free interval (DRFi) was the primary end-point.

Results

High expression of all investigated PDGF family members correlated to increasing Nottingham histopathological grade and high Ki67. Tumour cells displayed high expression of PDGFRα in 20%, and PDGF-CC in 21% of primary tumours, which correlated with the triple-negative subtype (TNBC). Patients with high PDGF-CC had inferior prognosis (P = 0.04) in terms of 5-year DRFi, whereas PDGFRα was up-regulated in lymph node metastasis and recurrences compared to primary tumours. High primary tumour PDGFRα was associated with increased risk of central nervous system (CNS) recurrence.

Conclusions

High PDGFRα and PDGF-CC expression were linked to breast cancer with an aggressive biological phenotype, e.g. the TNBC subtype, and high PDGF-CC increased the risk of 5-year distant recurrence. Tumour cell PDGFRα was significantly up-regulated in lymph node metastases and asynchronous recurrences. Our findings support an active role of the PDGF signalling pathway in tumour progression.

     

WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy

WD-repeat proteins are regulatory beta-propeller platforms that enable the assembly of multiprotein complexes. Here, we report the functional and bioinformatic analysis of human WD-repeat protein Interacting with PhosphoInosides (WIPI)-1alpha (WIPI49/Atg18), a member of a novel WD-repeat protein family with autophagic capacity in Saccharomyces cerevisiae and Caenorhabditis elegans, recently identified as phospholipid-binding effectors. Our phylogenetic analysis divides the WIPI protein family into two paralogous groups that fold into 7-bladed beta-propellers. Structural modeling identified two evolutionary conserved interaction sites in WIPI propellers, one of which may bind phospholipids. Human WIPI-1alpha has LXXLL signature motifs for nuclear receptor interactions and binds androgen and estrogen receptors in vitro. Strikingly, human WIPI genes were found aberrantly expressed in a variety of matched tumor tissues including kidney, pancreatic and skin cancer. We found that endogenous hWIPI-1 protein colocalizes in part with the autophagosomal marker LC3 at punctate cytoplasmic structures in human melanoma cells. In addition, hWIPI-1 accumulated in large vesicular and cup-shaped structures in the cytoplasm when autophagy was induced by amino-acid deprivation. These cytoplasmic formations were blocked by wortmannin, a classic inhibitor of PI-3 kinase-mediated autophagy. Our data suggest that WIPI proteins share an evolutionary conserved function in autophagy and that autophagic capacity may be compromised in human cancers.     

Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype

In neuroblastoma specimens, HIF-2alpha but not HIF-1alpha is strongly expressed in well-vascularized areas. In vitro, HIF-2alpha protein was stabilized at 5% O2 (resembling end capillary oxygen conditions) and, in contrast to the low HIF-1alpha activity at this oxygen level, actively transcribed genes like VEGF. Under hypoxia (1% O2), HIF-1alpha was transiently stabilized and primarily mediated acute responses, whereas HIF-2alpha protein gradually accumulated and governed prolonged hypoxic gene activation. Knockdown of HIF-2alpha reduced growth of neuroblastoma tumors in athymic mice. Furthermore, high HIF-2alpha protein levels were correlated with advanced clinical stage and high VEGF expression and predicted poor prognosis in a clinical neuroblastoma material. Our results demonstrate the relevance of HIF-2alpha in neuroblastoma progression and have general tumor biological implications.