Studies show that elevated IGF-1 levels are associated with an increased risk of breast cancer; however, mechanisms through which IGF-1 promotes mammary tumorigenesis in vivo have not been fully elucidated. To assess the possible involvement of COX-2 signaling in the protumorigenic effects of IGF-1 in mammary glands, we used the unique BK5.IGF-1 mouse model in which transgenic (Tg) mice have significantly increased incidence of spontaneous and DMBA–induced mammary cancer compared to wild type (WT) littermates. Studies revealed that COX-2 expression was significantly increased in Tg mammary glands and tumors, compared to age-matched WTs. Consistent with this, PGE2 levels were also increased in Tg mammary glands. Analysis of expression of the EP receptors that mediate the effects of PGE2 showed that among the four G-protein-coupled receptors, EP3 expression was elevated in Tg glands. Up-regulation of the COX-2/PGE2/EP3 pathway was accompanied by increased expression of VEGF and a striking enhancement of angiogenesis in IGF-1 Tg mammary glands. Treatment with celecoxib, a selective COX-2 inhibitor, caused a 45% reduction in mammary PGE2 levels, attenuated the influx of mast cells and reduced vascularization in Tg glands. These findings indicate that the COX-2/PGE2/EP3 signaling pathway is involved in IGF-1–stimulated mammary tumorigenesis and that COX-2–selective inhibitors may be useful in the prevention or treatment of breast cancer associated with elevated IGF-1 levels in humans.
mammary cancer; IGF-1; COX-2; stroma; transgenic mouse
High levels of prostaglandin E2 (PGE2) synthesis resulting from the upregulation of COX-2 has been shown to be critical for the development of non-melanoma skin tumors. This effect of PGE2 is likely mediated by one or more of its 4 G-protein coupled membrane receptors, EP1–4. A previous study showed that BK5.EP1 transgenic mice produced more carcinomas than wild type (WT) mice using initiation/promotion protocols, although the tumor response was dependent on the type of tumor promoter used. In this study, a single topical application of either 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P), alone, was found to elicit squamous cell carcinomas (SCC) in the BK5.EP1 transgenic mice, but not in WT mice. While the epidermis of both WT and transgenic mice was hyperplastic several days after DMBA, this effect regressed in the WT mice while proliferation continued in the transgenic mice. Several parameters associated with carcinogen initiation were measured and were found to be similar between genotypes, including CYP1B1 and aromatase expression, B[a]P adduct formation, Ras activity and keratinocyte stem cell numbers. However, EP1 transgene expression elevated COX-2 levels in the epidermis and SCC could be completely prevented in DMBA-treated BK5.EP1 mice either by feeding the selective COX-2 inhibitor celecoxib in their diet or by crossing them onto a COX-2 null background. These data suggest that the tumor promoting/progressing effects of EP1 require the PGE2 synthesized by COX-2.
Prostaglandin E2; EP1 receptor; skin carcinogensis; COX-2
The current study was undertaken to investigate potential oncogenic functions of NanogP8, a tumor-specific retrogene homolog of Nanog (expressed in pluripotent cells), in transgenic animal models. To this end, human primary prostate tumor-derived NanogP8 was targeted to the cytokeratin 14 (K14) cellular compartment, and two lines of K14-NanogP8 mice were derived. The line 1 animals, expressing high levels of NanogP8, experienced perinatal lethality and developmental abnormalities in multiple organs, including the skin, tongue, eye, and thymus in surviving animals. On postnatal day 5 transgenic skin, for example, there was increased c-Myc expression and Ki-67+ cells accompanied by profound abnormalities in skin development such as thickened interfollicular epidermis and dermis and lack of hypodermis and sebaceous glands. The line 3 mice, expressing low levels of NanogP8, were grossly normal except cataract development by 4–6 mo of age. Surprisingly, both lines of mice do not develop spontaneous tumors related to transgene expression. Even more unexpectedly, high levels of NanogP8 expression in L1 mice actually inhibited tumor development in a two-stage chemical carcinogenesis model. Mechanistic studies revealed that constitutive NanogP8 overexpression in adult L1 mice reduced CD34+α6+ and Lrig-1+ bulge stem cells, impaired keratinocyte migration, and repressed the expression of many stem cell-associated genes, including Bmp5, Fgfr2, Jmjd1a, and Jun. Our study, for the first time, indicates that transgenically expressed human NanogP8 is biologically functional, but suggests that high levels of NanogP8 may disrupt normal developmental programs and inhibit tumor development by depleting stem cells.
NanogP8; stem cells; K14; epidermis; tumor development
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.
To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5′ handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.
archaea; Haloferax volcanii; CRISPR/Cas; crRNA; PAM; seed sequence
Gene knockout studies unexpectedly reveal a pivotal role for IκB kinase alpha (IKKα) in mouse embryonic skin development. Skin carcinogenesis experiments show that Ikkα heterozygous mice are highly susceptible to chemical carcinogen or ultraviolet B light (UVB) induced benign and malignant skin tumors in comparison to wild-type mice. IKKα deletion mediated by keratin 5 (K5).Cre or K15.Cre in keratinocytes induces epidermal hyperplasia and spontaneous skin squamous cell carcinomas (SCCs) in Ikkα floxed mice. On the other hand, transgenic mice overexpressing IKKα in the epidermis, under the control of a truncated loricrin promoter or K5 promoter, develop normal skin and show no defects in the formation of the epidermis and other epithelial organs, and the transgenic IKKα represses chemical carcinogen or UVB induced skin carcinogenesis. Moreover, IKKα deletion mediated by a mutation, which generates a stop codon in the Ikkα gene, has been reported in a human autosomal recessive lethal syndrome. Downregulated IKKα and Ikkα mutations and deletions are found in human skin SCCs. The collective evidence not only highlights the importance of IKKα in skin development, maintaining skin homeostasis, and preventing skin carcinogenesis, but also demonstrates that mouse models are extremely valuable tools for revealing the mechanisms underlying these biological events, leading our studies from bench side to bedside.
skin carcinogenesis; skin development; IKKalpha (IKKα)
Although a major mechanism for cardioprotection is altered metabolism, little is known regarding metabolic changes in ischaemic preconditioning and subsequent ischaemia. Our objective was to examine the effects of the second window of preconditioning (SWOP), the delayed phase of preconditioning against infarction and stunning, on long-chain free fatty acid (LCFA) oxidation during ischaemia in chronically instrumented, conscious pigs.
Methods and results
We studied three groups: (i) normal baseline perfusion (n = 5); (ii) coronary artery stenosis (CAS; n = 5); (iii) CAS 24 h following 2 × 10 min coronary occlusions and 10 min reperfusion (n = 7). Ischaemia was induced by a left anterior descending (LAD) stenosis (40% flow reduction) for 90 min, dropping systolic wall thickening by 72%. LCFA oxidation was assessed following LAD infusion of 13C palmitate, i.e. during control or stenosis, by in vitro nuclear magnetic resonance of the sampled myocardium. Stenosis reduced subendocardial blood flow subendocardially, but not subepicardial, yet induced transmural reductions in LCFA oxidation and increased non-oxidative glycolysis. During stenosis, preconditioned hearts showed normalized contributions of LCFA to oxidative ATP synthesis, despite increased lactate accumulation. SWOP induced a shift towards LCFA oxidation during stenosis, despite increased malonyl-CoA, and marked protection of contractile function with a significant improvement in systolic wall thickening.
Thus, the second window of preconditioning normalized oxidative metabolism of LCFA during subsequent ischaemia despite elevated non-oxidative glycolysis and malonyl-CoA and was linked to protection of regional contractile function resulting in improved mechanical performance. Interestingly, the metabolic responses occurred transmurally while ischaemia was restricted solely to the subendocardium.
Mitochondria; Coronary stenosis; Long-chain fatty acids; β-Oxidation; Ischaemic preconditioning
Lysosomal cysteine protease cathepsin L (CTSL) is believed to play a role in tumor progression and is considered a marker for clinically invasive tumors. Studies from our laboratory using the classical mouse skin carcinogenesis model, with 7,12-dimethyl-benz[a]anthracene (DMBA) for initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) for promotion, showed that expression of CTSL is increased in papillomas and squamous cell carcinomas (SCC). We also carried out carcinogenesis studies using Ctsl-deficient nackt (nkt) mutant mice on three different inbred backgrounds. Unexpectedly, the multiplicity of papillomas were significantly higher in Ctsl-deficient than in wild-type mice on two unrelated backgrounds. Topical applications of TPA or DMBA alone to the skin of nkt/nkt mice did not induce papillomas, and there was no increase in spontaneous tumors in nkt/nkt mice on any of the three inbred backgrounds. Reduced epidermal cell proliferation in Ctsl-deficient nkt/nkt mice after TPA treatment suggested that they are not more sensitive than wild-type mice to TPA promotion. We also showed that deficiency of CTSL delays terminal differentiation of keratinocytes, and we propose that decreased elimination of initiated cells is at least partially responsible for the increased papilloma formation in the nackt model.
mouse models; proteases; cysteine cathepsins; skin cancer; two-stage carcinogenesis
This article endeavors to evaluate the data on the efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) and Coxibs in preclinical studies on cancer prevention carried out by the authors. The overall objective was to address questions that we see as significant for the field. The preclinical studies evaluated here are restricted to our rodent studies on colon/intestinal, bladder and non-melanoma skin cancer in which NSAIDs or celecoxib were administered as either prevention agents or therapeutic agents. These studies may shed light on several questions. Should human use of NSAIDs/Coxibs consider not only efficacy but also whether celecoxib is unique compared to other NSAIDs? Are standard NSAIDs as effective as celecoxib in animal studies? Is the efficacy of celecoxib in particular or NSAIDs in general due to their off-target effects or to their effects on COX-1 and COX-2? What is the likely efficacy of low dose aspirin? Some questions raised by human trials and human epidemiology are discussed and related to our observations in animal models. We also considered the problem with cardiovascular (CV) events and whether animal models are predictive of efficacy in humans. Based on human epidemiological studies and its CV profile, it appears that aspirin is the most promising NSAID for the prevention of human colon, bladder and skin cancer, although the animal data for aspirin is less conclusive. We hope that this discussion of the results in animal studies may help inform and shape human trials of these commonly employed, relatively inexpensive and highly effective classes of compounds.
chemoprevention; prostaglandins; COX-2; NSAIDs
Prokaryotes have developed several strategies to defend themselves against foreign genetic elements. One of those defense mechanisms is the recently identified CRISPR/Cas system, which is used by approximately half of all bacterial and almost all archaeal organisms. The CRISPR/Cas system differs from the other defense strategies because it is adaptive, hereditary and it recognizes the invader by a sequence specific mechanism. To identify the invading foreign nucleic acid, a crRNA that matches the invader DNA is required, as well as a short sequence motif called protospacer adjacent motif (PAM). We recently identified the PAM sequences for the halophilic archaeon Haloferax volcanii, and found that several motifs were active in triggering the defense reaction. In contrast, selection of protospacers from the invader seems to be based on fewer PAM sequences, as evidenced by comparative sequence data. This suggests that the selection of protospacers has stricter requirements than the defense reaction. Comparison of CRISPR-repeat sequences carried by sequenced haloarchaea revealed that in more than half of the species, the repeat sequence is conserved and that they have the same CRISPR/Cas type.
Haloferax volcanii; CRISPR/Cas; PAM; archaea; prokaryotic immune system; haloarchaea
Changes in metabolic and myofilament phenotypes coincide in developing hearts. Posttranslational modification of sarcomere proteins influences contractility, affecting the energetic cost of contraction. However, metabolic adaptations to sarcomeric phenotypes are not well understood, particularly during pathophysiological stress. This study explored metabolic adaptations to expression of the fetal, slow skeletal muscle troponin I (ssTnI). Hearts expressing ssTnI exhibited no significant ATP loss during 5 minutes of global ischemia, while non-transgenic littermates (NTG) showed continual ATP loss. At 7 min ischemia TG-ssTnI hearts retained 80±12% of ATP vs. 49±6% in NTG (P<0.05). Hearts expressing ssTnI also had increased AMPK phosphorylation. The mechanism of ATP preservation was augmented glycolysis. Glycolytic end products (lactate and alanine) were 38% higher in TG-ssTnI than NTG at 2 min and 27% higher at 5 min. This additional glycolysis was supported exclusively by exogenous glucose, and not glycogen. Thus, expression of a fetal myofilament protein in adult mouse hearts induced elevated anaerobic ATP production during ischemia via metabolic adaptations consistent with the resistance to hypoxia of fetal hearts. The general findings hold important relevance to both our current understanding of the association between metabolic and contractile phenotypes and the potential for invoking cardioprotective mechanisms against ischemic stress.
Troponin; Glycolysis; Adenosine Triphosphate (ATP); AMP Activated Protein Kinase, (AMPK); NMR Spectroscopy
Non-coding RNAs are key players in many cellular processes within organisms from all three domains of life. The range and diversity of small RNA functions beyond their involvement in translation and RNA processing was first recognized for eukaryotes and bacteria. Since then, small RNAs were also found to be abundant in archaea. Their functions include the regulation of gene expression and the establishment of immunity against invading mobile genetic elements. This review summarizes our current knowledge about small RNAs used for regulation and defence in archaea.
sRNA; Lsm; Hfq; Archaea; CRISPR; crRNA
Background: CRISPR/Cas systems allow archaea and bacteria to resist invasion by foreign nucleic acids.
Results: The CRISPR/Cas system in Haloferax recognized six different PAM sequences that could trigger a defense response.
Conclusion: The PAM sequence specificity of the defense response in type I CRISPR systems is more relaxed than previously thought.
Significance: The PAM sequence requirements for interference and adaptation appear to differ markedly.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.
Archaea; Microbiology; RNA; RNA Metabolism; RNA Processing; CRISPR/Cas; Haloferax volcanii; PAM
Risk of pancreatic cancer, the fourth deadliest cancer in the U.S., is increased by obesity. Calorie restriction (CR) prevents obesity, suppresses carcinogenesis in many models, and reduces serum levels of insulin-like growth factor (IGF)-1. In the present study, we examined the impact of CR on a model of inflammation-associated pancreatitis and pancreatic dysplasia, with a focus on the mechanistic contribution of systemic IGF-1. Administration of a 30% CR diet for 14 weeks decreased serum IGF-1 and hindered pancreatic ductal lesion formation and dysplastic severity, relative to a higher calorie control diet, in transgenic mice overexpressing cyclooxygenase (COX)-2 (BK5.COX-2). These findings in CR mice correlated with reductions in Ki-67-positive cells, vascular luminal size, vascular endothelial growth factor expression, and phosphorylation and total expression of downstream mediators of the IGF-1 pathway. Cell lines derived from BK5.COX-2 ductal lesions (JC101cells) formed pancreatic tumors in wild-type FVB mice that were significantly reduced in size by a 14-week CR regimen, relative to the control diet. To further understand the impact of circulating levels of IGF-1 on tumor growth in this model, we orthotopically injected JC101 cells into liver-specific IGF-1-deficient (LID) mice. The ~65% reduction of serum IGF-1 in LID mice resulted in significantly decreased burden of JC101 tumors, despite modestly elevated levels of circulating insulin and leptin. These data show that CR prevents development of dysplasia and growth of pancreatic cancer through alterations in IGF-1, suggesting that modulation of this pathway with dietary and/or pharmacologic interventions is a promising pancreatic cancer prevention strategy.
Pancreatic cancer; Calorie restriction; Insulin-like growth factor-1; Energy balance; pancreatitis; BK5.COX-2
Etiologic factors for pancreatic cancer, the fourth deadliest malignant neoplasm in the United States, include obesity and abnormal glucose metabolism. Calorie restriction (CR) and rapamycin each affect energy metabolism and cell survival pathways via inhibition of mammalian target of rapamycin (mTOR) signaling. Using a Panc02 murine pancreatic cancer cell transplant model in 45 male C57BL/6 mice, we tested the hypothesis that rapamycin mimics the effects of CR on pancreatic tumor growth. A chronic regimen of CR, relative to an ad libitum-fed control diet, produced global metabolic effects such as reduced body weight (20.6±1.6g vs. 29.3±2.3g; p<0.0001), improved glucose responsiveness, and decreased circulating levels of insulin-like growth factor (IGF)-1 (126±8ng/mL vs. 199±11ng/mL; p=0.0006) and leptin (1.14±0.2 ng/mL vs. 5.05±1.2 ng/mL; p=0.01). In contrast, rapamycin treatment (2.5mg/kg i.p. every other day, initiated in mice following 20 weeks of ad libitum control diet consumption), relative to control diet, produced no significant change in body weight, IGF-1 or leptin levels, but decreased glucose responsiveness. Pancreatic tumor volume was significantly reduced in the CR group (221±107mm3; p<0.001) and, to a lesser extent, the rapamycin group (374±206mm3; p=0.04) relative to controls (550±147mm3), and this differential inhibition correlated with expression of the proliferation marker Ki-67. Both CR and rapamycin decreased phosphorylation of mTOR, p70/S6K and S6 ribosomal protein, but only CR decreased phosphorylation of Akt, GSK-3β, ERK/MAPK, and STAT-3TYR705. These findings suggest rapamycin partially mimics the anticancer effects of calorie restriction on tumor growth in a murine model of pancreatic cancer.
Pancreatic cancer; Calorie restriction; Rapamycin; Insulin-like growth factor-1; IGF-1; Energy balance; mammalian target of rapamycin; mTOR
Testosterone is necessary for the development of male pattern baldness, known as androgenetic alopecia (AGA); yet, the mechanisms for decreased hair growth in this disorder are unclear. We show that prostaglandin D2 synthase (PTGDS) is elevated at the mRNA and protein levels in bald scalp compared to haired scalp of men with AGA. The product of PTGDS enzyme activity, prostaglandin D2 (PGD2), is similarly elevated in bald scalp. During normal follicle cycling in mice, Ptgds and PGD2 levels increase immediately preceding the regression phase, suggesting an inhibitory effect on hair growth. We show that PGD2 inhibits hair growth in explanted human hair follicles and when applied topically to mice. Hair growth inhibition requires the PGD2 receptor G protein (heterotrimeric guanine nucleotide)–coupled receptor 44 (GPR44), but not the PGD2 receptor 1 (PTGDR). Furthermore, we find that a transgenic mouse, K14-Ptgs2, which targets prostaglandin-endoperoxide synthase 2 expression to the skin, demonstrates elevated levels of PGD2 in the skin and develops alopecia, follicular miniaturization, and sebaceous gland hyperplasia, which are all hallmarks of human AGA. These results define PGD2 as an inhibitor of hair growth in AGA and suggest the PGD2-GPR44 pathway as a potential target for treatment.
The metallo-β-lactamase family of enzymes comprises a large group of proteins with diverse functions in the metabolism of the cell. Among others, this superfamily contains proteins which are involved in DNA and RNA metabolism, acting as nucleases in e.g. repair and maturation. Many proteins have been annotated in prokaryotic genomes as being potential metallo-β-lactamases, but very often the function has not been proven. The protein HVO_2763 from Haloferax volcanii is such a potential metallo-β-lactamase. HVO_2763 has sequence similarity to the metallo-β-lactamase tRNase Z, a tRNA 3′ processing endonuclease. Here, we report the characterisation of this metallo-β-lactamase HVO_2763 in the halophilic archaeon Haloferax volcanii. Using different in vitro assays with the recombinant HVO_2763, we could show that the protein does not have tRNA 3′ processing or exonuclease activity. According to transcriptome analyses of the HVO_2763 deletion strain, expression of proteins involved in membrane transport is downregulated in the mutant. Therefore, HVO_2763 might be involved directly or indirectly in membrane transport.
Electronic supplementary material
The online version of this article (doi:10.1007/s00792-012-0433-4) contains supplementary material, which is available to authorized users.
Metallo-β-lactamase; tRNase Z; Haloferax volcanii; PhnP; HVO_2763
Many tumor cells have elevated rates of glucose uptake that can be measured quantitatively, noninvasively and repeatedly by positron emission tomography (PET) with 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG). Clinical imaging with 18F-FDG PET has been used for detection and staging of primary and metastatic tumors. High-resolution microPET scanning and murine cancer models make it possible to analyze longitudinally glucose metabolism during the appearance, development and progression of individual experimental tumors. In this study, we used 18F-FDG microPET and micro computerized tomography (microCT) to investigate glucose uptake in the DMBA/TPA chemically-induced multistage mouse skin carcinogenesis model. 18F-FDG uptake is significantly higher in all papillomas than in surrounding skin. Elevated 18F-FDG uptake is observed when tumors can be identified morphologically, but not before. Although 18F-FDG uptake is high in all fully invasive, malignant skin squamous cell carcinomas, uptake in papillomas and microinvasive malignant squamous cell carcinomas is variable and does not exhibit any correlation with tumor stage.
glucose metabolism; PET; skin cancer; molecular imaging; fluorodeoxyglucose
In this report, we describe the development of a transgenic mouse in which a rat probasin promoter (ARR2Pb) was used to direct prostate specific expression of a constitutively active form of signal transducer and activator of transcription 3 (i.e., Stat3C). ARR2Pb.Stat3C mice exhibited hyperplasia and prostate intraepithelial neoplasia (PIN) lesions in both ventral and dorsolateral prostate lobes at 6 and 12 months; however, no adenocarcinomas were detected. The effect of combined loss of PTEN was examined by crossing ARR2Pb.Stat3C mice with PTEN+/- null mice. PTEN+/- null mice on an ICR genetic background developed only hyperplasia and PIN at 6 and 12 months, respectively. ARR2Pb.Stat3C x PTEN+/- mice exhibited a more severe prostate phenotype compared with ARR2Pb.Stat3C and PTEN+/- mice. ARR2Pb.Stat3C x PTEN+/- mice developed adenocarcinomas in the ventral prostate as early as 6 months (22% incidence) that reached an incidence of 61% by 12 months. Further evaluations indicated that phospho-Stat3, phospho-Akt, phospho-nuclear factor κB, cyclin D1, and Ki67 were upregulated in adenocarcinomas from ARR2Pb.Stat3C x PTEN+/- mice. In addition, membrane staining for β-catenin and E-cadherin was reduced. The changes in Stat3 and nuclear factor κB phosphorylation correlated most closely with tumor progression. Collectively, these data provide evidence that Stat3 and Akt signaling cooperate in prostate cancer development and progression and that ARR2Pb.Stat3C x PTEN+/- mice represent a novel mouse model of prostate cancer to study these interactions.
Multiple molecular mechanisms are involved in the promotion of skin carcinogenesis. Induction of sustained proliferation and epidermal hyperplasia by direct activation of mitotic signaling pathways or indirectly in response to chronic wounding and/or inflammation, or due to a block in terminal differentiation or resistance to apoptosis is necessary to allow clonal expansion of initiated cells with DNA mutations to form skin tumors. The mitotic pathways include activation of epidermal growth factor receptor and Ras/Raf/mitogen-activated protein kinase signaling. Chronic inflammation results in inflammatory cell secretion of growth factors and cytokines such as tumor necrosis factor-a and interleukins, as well as production of reactive oxygen species, all of which can stimulate proliferation. Persistent activation of these pathways leads to tumor promotion.
skin carcinogenesis; tumor promoters; 12-O-tetradecanoylphorbol 13-acetate (TPA); protein kinase C (PKC); epidermal growth factor receptor (EGFR); transforming growth factor-β (TGFβ); tumor necrosis factor-α (TNFα); interleukins; cyclooxygenase-2 (COX-2); ornithine decarboxylase (ODC)
Peroxisome proliferator–activated receptor-delta (PPAR-δ) is overexpressed in human colon cancer, but its contribution to colonic tumorigenesis is controversial. We generated a mouse model in which PPAR-δ was genetically disrupted in colonic epithelial cells by targeted deletion of exon 4. Elimination of colon-specific PPAR-δ 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-δ genetic disruption (10–11 mice per group) were tested for incidence of azoxymethane-induced colon tumors. The effects of targeted PPAR-δ deletion on vascular endothelial growth factor expression were determined by real-time RT-PCR. Targeted PPAR-δ genetic disruption inhibited colonic carcinogenesis: Mice with PPAR-δ(−/−) colons developed 98.5% fewer tumors than wild-type mice (PPAR-δ(−/−) 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-δ expression. These findings indicate that PPAR-δ has a crucial role in promoting colonic tumorigenesis.
Expression of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) inhibits gastrointestinal tumorigenesis in NAG-1 transgenic mice (C57/BL6 background). In the present study, we investigated whether NAG-1 protein would alter urethane-induced pulmonary lesions in NAG-1 transgenic mice on an FVB background (NAG-1Tg+/FVB). NAG-1Tg+/FVB mice had both decreased number and size of urethane-induced tumors, compared to control littermates (NAG-1Tg+/FVB = 16 ± 4 per mouse versus control = 20 ± 7 per mouse, p<0.05). Urethane-induced pulmonary adenomas (PAs) and adenocarcinomas (PACs) were observed in control mice, but only PAs were observed in NAG-1Tg+/FVB mice. Urethane-induced tumors from control littermates and NAG-1Tg+/FVB mice highly expressed proteins in the arachidonic acid pathway (cyclooxygenases 1/2, prostaglandin E synthase, and prostaglandin E2 receptor) and highly activated several kinases (phospho-Raf-1 and phospho-ERK1/2). However, only urethane-induced p38 MAPK phosphorylation was decreased in NAG-1Tg+/FVB mice. Furthermore, significantly increased apoptosis in tumors of NAG-1Tg+/FVB mice compared to control mice was observed as assessed by caspase 3/7 activity. In addition, fewer inflammatory cells were observed in the lung tissue isolated from urethane-treated NAG-1Tg+/FVB mice compared to control mice. These results paralleled in vitro assays using human A549 pulmonary carcinoma cells. Less phosphorylated p38 MAPK was observed in cells over-expressing NAG-1, compared to control cells. Overall, our study revealed for the first time that NAG-1 protein inhibits urethane-induced tumor formation, probably mediated by the p38 MAPK pathway, and is a possible new target for lung cancer chemoprevention.
NAG-1; lung tumorigenesis; p38 MAPK; apoptosis; inflammation
Multiple molecular mechanisms are involved in the promotion of skin carcinogenesis. Induction of sustained proliferation and epidermal hyperplasia by direct activation of mitotic signaling pathways or indirectly in response to chronic wounding and/or inflammation, or due to a block in terminal differentiation or resistance to apoptosis is necessary to allow clonal expansion of initiated cells with DNA mutations to form skin tumors. The mitotic pathways include activation of epidermal growth factor receptor and Ras/Raf/mitogen-activated protein kinase signaling. Chronic inflammation results in inflammatory cell secretion of growth factors and cytokines such as tumor necrosis factor-α and interleukins, as well as production of reactive oxygen species, all of which can stimulate proliferation. Persistent activation of these pathways leads to tumor promotion.
skin carcinogenesis; tumor promoters; 12-O-tetradecanoylphorbol 13-acetate (TPA); protein kinase C (PKC); epidermal growth factor receptor (EGFR); transforming growth factor-β (TGFβ); tumor necrosis factor-α (TNFα); interleukins; cyclooxygenase-2 (COX-2); ornithine decarboxylase (ODC)
Although prostaglandin E2 (PGE2) has been shown by pharmacological and genetic studies to be important in skin cancer, the molecular mechanism(s) by which it contributes to tumor growth is not well understood. In this study we investigated the mechanisms by which PGE2 stimulates murine keratinocyte proliferation using in vitro and in vivo models. In primary mouse keratinocyte (PMK) cultures, PGE2 activated the epidermal growth factor receptor (EGFR) and its downstream signaling pathways as well as increased cyclic AMP (cAMP) production and activated the cAMP response element binding protein (CREB). EGFR activation was not significantly inhibited by pretreatment with a c-src inhibitor (PP2), nor by a protein kinase A inhibitor (H-89). However, PGE2-stimulated extracellularly-regulated kinase1/2 (ERK1/2) activation was completely blocked by EGFR, ERK1/2 and phosphatidylinositol 3-kinase (PI3-K) pathway inhibitors. In addition, these inhibitors attenuated the PGE2-induced proliferation, nuclear factor-κB (NF-κB), activator protein-1 (AP-1) and CREB binding to the promoter regions of the cyclin D1 and vascular endothelial growth factor (VEGF) genes and expression of cyclin D1 and VEGF in PMKs. Similarly, in vivo, we found that wild type (WT) mice treated with PGE2 and untreated COX-2 overexpressing transgenic mice had higher levels of cell proliferation and expression of cyclin D1 and VEGF, as well as higher levels of activated EGFR, NF-κB, AP-1 and CREB, than vehicle-treated WT mice. Our findings provide evidence for a link between COX-2 overexpression and EGFR-, ERK-, PI3-K-, cAMP-mediated cell proliferation, and the tumor promoting activity of PGE2 in mouse skin.
A large body of studies has suggested that peroxisome proliferator-activated receptor γ (PPARγ) ligands, such as thiazolidinedione, are potent candidates for chemopreventive agents. MCC-555 is a PPARγ/α dual agonist and has been previously shown to induce apoptosis in vitro; however, the molecular mechanisms by which MCC-555 affects anti-tumorigenesis in vivo are poorly understood. In this study, we explored the anti-tumorigenic effects of MCC-555 both in cell culture and in Apc-deficient mice, an animal model for human familial adenomatous polyposis. MCC-555 increased MUC2 expression in colorectal and lung cancer cells, and treatment with the PPARγ antagonist GW9662 revealed that MUC2 induction by MCC-555 was mediated in a PPARγ-dependent manner. Moreover, MCC-555 increased transcriptional activity of human and mouse MUC2 promoters. Subsequently, treatment with MCC-555 (30 mg/kg/day) for 4 weeks reduced the number of small intestinal polyps to 54.8% of that in control mice. In agreement with in vitro studies, enhanced Muc2 expression was observed in the small intestinal tumors of Min mice treated with MCC-555, suggesting that MUC2 expression may be associated at least in part with the anti-tumorigenic action of MCC-555. In addition, highly phosphorylated extracellular signal-regulated kinase (ERK) was found in the intestinal tumors of MCC-555-treated Min mice, and inhibition of the ERK pathway by a specific inhibitor markedly suppressed MCC-555-induced Muc2 expression in vitro. Overall, these results indicate that MCC-555 has a potent tumor suppressor activity in intestinal tumorigenesis, likely involving MUC2 up-regulation by ERK and PPARγ pathways.
MCC-555; colorectal cancer; ApcMin/+ mice; MUC2; PPARs; ERK pathway