Carcinogenesis - current issue

Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics

Imai, K., Yamamoto, H. — 2008-04-01

DNA mismatch repair (MMR) deficiency results in a strong mutator phenotype and high-frequency microsatellite instability (MSI-H), which are the hallmarks of tumors arising within Lynch syndrome. MSI-H is characterized by length alterations within simple repeated sequences, microsatellites. Lynch syndrome is primarily due to germline mutations in one of the DNA MMR genes; mainly hMLH1 or hMSH2 and less frequently hMSH6 and rarely hPMS2. Germline hemiallelic methylation of MLH1, termed epimutation, has been reported to be a new cause of Lynch syndrome. MSI-H is also observed in ~15% of colorectal, gastric and endometrial cancers and in lower frequencies in a minority of other tumors, where it is associated with the hypermethylation of the promoter region of hMLH1. MSI-H underlies a distinctive tumorigenic pathway because cancers with MSI-H exhibit many differences in genotype and phenotype relative to cancers without MSI-H, irrespective of their hereditary or sporadic origins. Genetic, epigenetic and transcriptomic differences exist between cancers with and those without the MSI-H. The BRAF V600E mutation is associated with sporadic MSI-H colorectal cancers (CRCs) harboring hMLH1 methylation but not Lynch syndrome-related CRCs. The differences in genotype and phenotype between cancers with and those without MSI-H are likely to be causally linked to their differences in biological and clinical features. Therefore, the diagnosis of MSI-H in cancers is thus considered to be of increasing relevance, because MSI-H is a useful screening marker for identifying patients with Lynch syndrome, a better prognostic factor and could affect the efficacy of chemotherapy. This review addresses recent advances in the field of microsatellite instability research.

Detection of chromosome changes in pathology archives: an application of microwave-assisted fluorescence in situ hybridization to human carcinogenesis studies

Sugimura, H. — 2008-04-01

Pathology archives provide unique and abundant opportunities to investigate human carcinogenesis and identify potential targets for cancer therapy. Microwaving was introduced into various procedures used in histopathology two decades ago, although the precise mechanisms underlying its effectiveness in any of the procedures, including antigen retrieval, acceleration of fixation and nucleic acid hybridization, are not known. Since microwaving was first applied to fluorescence in situ hybridization (FISH), many pathologists and researchers have enjoyed the benefits of excellent preservation of histological structures as well as good retrieval of FISH signals by this method. Microwave-assisted fluorescence in situ hybridization (MW-FISH) has proved to be especially useful in retrospective investigations of tissues fixed and preserved for long periods of time, and the success rates in the randomly selected pathology archives have been greater (70–95%) than by the conventional protocol (≤40%) The MW-FISH protocol and current availability of human genome information together with information on a variety of other histopathological attributes have paved the way to exploration of specific, large-scale genomic changes in human tumor tissue, even in the incipient stage. In practice, this protocol is very useful for retrospective surveillance of amplicons in tumor tissue by using hundreds of bacterial artificial chromosome clones and many specimens in the form of a tissue microarray. Effective retrieval of specific genome-wide amplicon profiles from human tumors stored unaware in ordinary pathology laboratories would help to further stratify tumors so that individually tailored treatment strategies would become feasible in clinical settings.

15-Deoxy-{Delta}12,14-prostaglandin J2 induces COX-2 expression through Akt-driven AP-1 activation in human breast cancer cells: a potential role of ROS

2008-04-01

Recent studies suggest that inflammation is causally linked to carcinogenesis. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandins, is inappropriately expressed in various cancers and hence recognized as one of the hallmarks of chronic inflammation-associated malignancies. However, the mechanistic role of COX-2 as a link between inflammation and cancer remains undefined. Here, we report that 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂), one of the final products of COX-mediated arachidonic acid metabolism, upregulates the expression of COX-2 in the human breast cancer MCF-7 cell line. 15d-PGJ₂-induced COX-2 expression was mediated by activation of Akt and subsequently activator protein-1 (AP-1). Furthermore, 15d-PGJ₂ formed reactive oxygen species, which led to increased phosphorylation of Akt, DNA binding of AP-1 and expression of COX-2. In contrast to 15d-PGJ₂, 9,10-dihydro-15d-PGJ₂ did not elicit any of effects induced by 15d-PGJ₂ in this study, suggesting that an electrophilic carbon center present in 15d-PGJ₂ is critical for COX-2 expression as well activation of upstream signal transduction induced by this cyclopentenone prostaglandin. Taken together, these observations suggest that 15d-PGJ₂ produced by COX-2 overexpression may function as a positive regulator of COX-2 in human breast cancer MCF-7 cells.

Cyr61/CCN1 and CTGF/CCN2 mediate the proangiogenic activity of VHL-mutant renal carcinoma cells

2008-04-01

The von Hippel–Lindau (VHL) protein serves as a negative regulator of hypoxia-inducible factor (HIF)- subunits. Since HIF regulates critical angiogenic factors such as vascular endothelial growth factor (VEGF) and lesions in VHL gene are present in a majority of the highly vascularized renal cell carcinoma (RCC), it is believed that deregulation of the VHL–HIF pathway is crucial for the proangiogenic activity of RCC. Although VEGF has been confirmed as a critical angiogenic factor upregulated in VHL-mutant cells, the efficacy of antiangiogenic therapy specifically targeting VEGF signaling remains modest. In this study, we developed a three-dimensional in vitro assay to evaluate the ability of RCC cells to promote cord formation by the primary human dermal microvascular endothelial cells (HDMECs). Compared with VHL wild-type cells, VHL-mutant RCC cells demonstrated a significantly increased proangiogenic activity, which correlated with increased secretion of cysteine-rich 61 (Cyr61)/cysteine-rich 61-connective tissue growth factor-nephroblastoma overexpressed (CCN) 1, connective tissue growth factor (CTGF)/CCN2 and VEGF in conditioned culture medium. Both CCN proteins are required for HDMEC cord formation as shown by RNA interference knockdown experiments. Importantly, the proangiogenic activities conferred by the CCN proteins and VEGF are additive, suggesting non-overlapping functions. Expression of the CCN proteins is at least partly dependent on the HIF-2 function, the dominant HIF- isoform expressed in RCC. Finally, immunohistochemical staining of Cyr61/CCN1 and CTGF/CCN2 in RCC tissue samples showed that increased expression of these proteins correlates with the loss of VHL protein expression. These findings strengthened the notion that the hypervascularized phenotype of RCC is afforded by multiple proangiogenic factors that function in parallel pathways.

Macrophage inhibitory cytokine-1 activates AKT and ERK-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells

Kim, K.-K., Lee, J. J., Yang, Y., You, K.-H., Lee, J.-H. — 2008-04-01

Macrophage inhibitory cytokine-1 (MIC-1) is a member of the transforming growth factor-β superfamily, which is overexpressed in a variety of human cancers, including breast and gastric cancer. The function of MIC-1 in cancer remains controversial and its signaling pathways remain poorly understood. In this study, we demonstrate that MIC-1 induces the transactivation of ErbB2 in SK-BR-3 breast and SNU-216 gastric cancer cells. MIC-1 induced a significant phosphorylation of Akt and ERK-1/2, and also effected an increase in the levels of tyrosine phosphorylation of ErbB1, ErbB2 and ErbB3 in SK-BR-3 and SNU-216 cells. The treatment of these cells with AG825 and AG1478, inhibitors specific for ErbB2 tyrosine kinase, resulted in the complete abolition of MIC-1-induced Akt and ERK-1/2 phosphorylation. Furthermore, the small-interfering RNA-mediated downregulation of ErbB2 significantly reduced not only the phosphorylation of Akt and ERK-1/2 but also the invasiveness of the cells induced by MIC-1. Our results show that ErbB2 activation performs a crucial function in MIC-1-induced signaling pathways. Further investigations revealed that MIC-1 induced the expression of the hypoxia inducible factor-1 protein and the expression of its target genes, including vascular endothelial growth factor, via the activation of the mammalian target of rapamycin (mTOR) signaling pathway. Stimulation of SK-BR-3 with MIC-1 profoundly induces the phosphorylation of mTOR and its downstream substrates, including p70S6K and 4E-BP1. Collectively, these results show that MIC-1 may participate in the malignant progression of certain human cancer cells that overexpress ErbB2 through the transactivation of ErbB2 tyrosine kinase.

Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMP-activated protein kinase in DU145 human prostate cancer cells

2008-04-01

Hypoxia-inducible factor (HIF-1) plays a central role in the cellular adaptive response to hypoxic conditions, which are closely related to pathophysiological conditions, such as cancer. Although reactive oxygen species (ROS) have been implicated in the regulation of hypoxic and non-hypoxic induction of HIF-1 under various conditions, the role of ROS is quite controversial, and the mechanism underlying the HIF-1 regulation by ROS is not completely understood yet. Here, we investigated the biochemical mechanism for the ROS-induced HIF-1 by revealing a novel role of adenosine monophosphate-activated protein kinase (AMPK) and the upstream signal components. AMPK plays an essential role as energy-sensor under adenosine triphosphate-deprived conditions. Here we report that ROS induced by a direct application of H₂O₂ and menadione to DU145 human prostate carcinoma resulted in accumulation of HIF-1 protein by attenuation of its degradation and activation of its transcriptional activity in an AMPK-dependent manner. By way of contrast, AMPK was required only for the transcriptional activity of HIF-1 under hypoxic condition, revealing a differential role of AMPK in these two stimuli. Furthermore, our data show that inhibition of AMPK enhances HIF-1 ubiquitination under ROS condition. Finally, we show that the regulation of HIF-1 by AMPK in response to ROS is under the control of c-Jun N-terminal kinase and Janus kinase 2 pathways. Collectively, our findings identify AMPK as a key determinant of HIF-1 functions in response to ROS and its possible role in the sophisticated HIF-1 regulatory mechanisms.

Cells deficient in oxidative DNA damage repair genes Myh and Ogg1 are sensitive to oxidants with increased G2/M arrest and multinucleation

2008-04-01

Oxidative stress generated from endogenous and exogenous sources causes oxidative DNA damage. The most frequent mutagenic base lesion 7,8-dihydro-8-oxoguanine and the resulting mismatched adenine are removed by OGG1 and MYH in mammals. Deficiencies in human MYH or mouse MYH and OGG1 result in tumor predisposition but the underlying molecular mechanism is not fully understood. To facilitate the study of the roles of MYH and OGG1 in the protection against oxidative stress, we generated mouse embryonic fibroblast cell lines deficient in these genes. Myh and Ogg1 double knockout cells were more sensitive than wild type to oxidants (hydrogen peroxide and t-butyl hydroperoxide), but not to cis-platinum or -irradiations. The low dosage oxidative stress resulted in more reduction of S phase and increase of G₂/M phase in Myh^–/–Ogg1^–/– cells than in wild-type cells, but a similar level of cell death in both cells. The oxidants also induced more multinucleated cells in Myh^–/–Ogg1^–/– cells than in wild-type, accompanied by centrosome amplification and multipolar spindle formation. Thus, under oxidative stress, Myh and Ogg1 are likely required for normal cell-cycle progression and nuclear division, suggesting multiple roles of Myh and Ogg1 in the maintenance of genome stability and tumor prevention.

Interactions of acetylcholinesterase with caveolin-1 and subsequently with cytochrome c are required for apoptosome formation

2008-04-01

Acetylcholinesterase (AChE) is emerging as an important component in leading to apoptosis. Our previous study demonstrated that silencing of the AChE gene blocked the interaction between cytochrome c and apoptotic protease-activating factor-1 (Apaf-1) in etoposide-induced apoptosis of HT-29 cells. We undertook this study to further dissect the molecular role of AChE in apoptosome formation. The present study elicited that small interfering RNA (siRNA) to cytochrome c gene blocked the interaction of AChE with Apaf-1, whereas siRNA to Apaf-1 gene did not block the interaction of AChE with cytochrome c, indicating that the interaction of AChE with cytochrome c is required for the interaction between cytochrome c and protease-activating factor-1. We further observed that AChE is localized to caveolae via interacting with caveolin-1 during apoptosis and that the disruption of caveolae prevented apoptosome formation. These data indicate that the interactions of AChE with caveolin-1 and subsequently with cytochrome c appear to be indispensable for apoptosome formation.

GLI1 repression of ERK activity correlates with colony formation and impaired migration in human epidermal keratinocytes

2008-04-01

Basal cell carcinoma (BCC) of the skin is a highly compact, non-metastatic epithelial tumour type that may arise from the aberrant propagation of epidermal or progenitor stem cell (SC) populations. Increased expression of GLI1 is a common feature of BCC and is linked to the induction of epidermal SC markers in immortalized N/Tert-1 keratinocytes. Here, we demonstrate that GLI1 over-expression is linked to additional SC characteristics in N/Tert-1 cells including reduced epidermal growth factor receptor (EGFR) expression and compact colony formation that is associated with repressed extracellular signal-regulated kinase (ERK) activity. Colony formation and repressed ERK activity remain evident when EGFR is increased exogenously to the basal levels in GLI1 cells revealing that ERK is additionally inhibited downstream of the receptor. Exposure to epidermal growth factor (EGF) to increase ERK activity and promote migration negates GLI1 colony formation with cells displaying an elongated, fibroblast-like morphology. However, as determined by Snail messenger RNA and E-cadherin protein expression this is not associated with epithelial–mesenchymal transition (EMT), and GLI1 actually represses induction of the EMT marker vimentin in EGF-stimulated cells. Instead, live cell imaging revealed that the elongated morphology of EGF/GLI1 keratinocytes stems from their being ‘stretched’ due to migrating cells displaying inefficient cell–cell detachment and impaired tail retraction. Taken together, these data suggest that GLI1 opposes EGFR signalling to maintain the epithelial phenotype. Finally, ERK activity was predominantly negative in 13/14 BCCs (superficial/nodular), indicating that GLI1 does not routinely co-operate with ERK to induce the formation of this common skin tumour.

Telomere dysfunction promotes genome instability and metastatic potential in a K-ras p53 mouse model of lung cancer

2008-04-01

Current mouse models of lung cancer recapitulate signature genetic lesions and some phenotypic features of human lung cancer. However, because mice have long telomeres, models to date do not recapitulate the aspects of lung carcinogenesis—telomere attrition and the genomic instability that ensues—believed to serve as key mechanisms driving lung tumor initiation and progression. To explore the contributions of telomere dysfunction to lung cancer progression, we combined a telomerase catalytic subunit (mTerc) mutation with the well-characterized K-rasG12D mouse lung cancer model. K-ras^G12D mTerc^–/– mice with telomere dysfunction but intact p53 exhibited increased lung epithelial apoptosis, delayed tumor formation and increased life span relative to K-ras^G12D mTerc^+/– mice with intact telomere function. This demonstrates that by itself, telomere dysfunction acts in a tumor-suppressive mechanism. Introduction of a heterozygous p53 mutation exerted a marked histopathological, biological and genomic impact. K-ras^G12D mTerc^–/– p53^+/– mice developed aggressive tumors with more chromosomal instabilities and high metastatic potential, leading to decreased overall survival. Thus, we have generated a murine model that more faithfully recapitulates key aspects of the human disease. Furthermore, these findings clearly demonstrate (in an in vivo model system) the dual nature of telomere shortening as both a tumor-suppressive and tumor-promoting mechanism in lung cancer, dependent on p53 status.

MDM2 SNP309 G allele increases risk but the T allele is associated with earlier onset age of sporadic breast cancers in the Chinese population

2008-04-01

Sporadic breast cancer in women <40 years is uncommon in Caucasians, in contrast to a much earlier onset in Chinese Asians. However, the molecular determinants for this earlier onset are unclear. It has been reported that SNP309 in the promoter of MDM2, the negative regulator of p53, affects the onset age of cancers in females. Essentially, the G allele, rather than the T allele, has been suggested to accelerate the age of cancer onset. Hence, we examined if MDM2 and p53 polymorphisms would be determinants of the early onset phenomenon in Chinese women. Our results indicate that the MDM2 SNP309 G allele is more prevalent in the Chinese population compared with reported frequencies in Caucasians, and increases breast cancer risk of both sporadic cases and those with family history. However, it was the T/T genotype that was associated with earlier onset age of sporadic breast cancers in contrast to the G allele that was associated with the familial cases. Though p53 codon 72 single-nucleotide polymorphism (SNP) did not affect general cancer risk or age of onset, arginine homozygozity, in contrast to proline homozygozity, was found to decrease breast cancer risk in the later onset sporadic cases. Both SNP309 and codon 72 polymorphisms did not affect the stage of cancer. Together, the data suggest that though the MDM2 SNP309 G allele is a risk factor for breast cancer, it does not accelerate, but delays the onset of the sporadic disease in Chinese women, highlighting that differences in ethnicity and family history may influence the role of MDM2 SNP309 in cancer susceptibility.

Constitutional CHEK2 mutations are associated with a decreased risk of lung and laryngeal cancers

2008-04-01

Mutations in the CHEK2 gene have been associated with increased risks of breast, prostate and colon cancer. In contrast, a previous report suggests that individuals with the I157T missense variant of the CHEK2 gene might be at decreased risk of lung cancer and upper aero-digestive cancers. To confirm this hypothesis, we genotyped 895 cases of lung cancer, 430 cases of laryngeal cancer and 6391 controls from Poland for four founder alleles in the CHEK2 gene, each of which has been associated with an increased risk of cancer at several sites. The presence of a CHEK2 mutation was protective against both lung cancer [odds ratio (OR) = 0.3; 95% confidence interval (CI) 0.2–0.5; P = 3 x 10^–8] and laryngeal cancer (OR = 0.6; 95% CI 0.3–0.99; P = 0.05). The basis of the protective effect is unknown, but may relate to the reduced viability of lung cancer cells with a CHEK2 mutation. Lung cancers frequently possess other defects in genes in the DNA damage response pathway (e.g. p53 mutations) and have a high level of genotoxic DNA damage induced by tobacco smoke. We speculate that lung cancer cells with impaired CHEK2 function undergo increased rates of cell death.

Estrogen-biosynthesis gene CYP17 and its interactions with reproductive, hormonal and lifestyle factors in breast cancer risk: results from the Long Island Breast Cancer Study Project

2008-04-01

The genes that are involved in estrogen biosynthesis, cellular binding and metabolism may contribute to breast cancer susceptibility. We examined the effect of the CYP17 promoter T -> C polymorphism and its interactions with the reproductive history, exogenous hormone use and selected lifestyle risk factors on breast cancer risk among 1037 population-based incident cases and 1096 population-based controls in the Long Island Breast Cancer Study Project. Overall, there were no associations between the CYP17 genotype and breast cancer risk. Among postmenopausal women, the joint exposure to higher body mass index (BMI) and the variant C allele was associated with an increased risk of breast cancer [odds ratio (OR), 1.60; 95% confidence interval (CI), 1.15–2.22]. The joint exposure to the variant C allele and long-term use of hormone replacement therapy (HRT) (>51 months) was related to an increased risk of breast cancer (OR, 1.51; 95% CI, 0.99–2.31) especially estrogen receptor-positive, progesterone receptor-positive breast cancer (OR, 1.87; 95% CI, 1.08–3.25). Among the control population, the CYP17 variant C allele was inversely associated with long-term use of postmenopausal HRT and a higher BMI in postmenopausal women. In conclusion, the findings suggest that the CYP17 variant C allele may increase breast cancer risk in conjunction with long-term HRT use and high BMI in postmenopausal women.

Comparison of induced and cancer-associated mutational spectra using multivariate data analysis

Lewis, P.D., Manshian, B., Routledge, M.N., Scott, G.B., Burns, P.A. — 2008-04-01

One of the most useful tools for investigating the aetiopathology of cancer is the mutation spectrum, which comprises the type and distribution of mutations within a gene sequence. Many studies have generated mutagen-induced spectra using in vitro or in vivo model systems in an attempt to find correlations with those observed in cancer-associated genes such as the TP53 tumour suppressor gene. Consequently, meaningful similarities in the types of mutation found in induced and human spectra have been demonstrated. However, it is more difficult to draw such conclusions about the distribution or sequence context of mutations when they arise in different target sequences. We have developed an analytical approach for base substitution spectra that capture information for both sequence context and mutation type simultaneously. The resulting mutation signature is a fixed set of data points that allows comparison of multiple mutation spectra regardless of sequence. We have applied this method to a mixed set of mutation spectra observed in exons 5, 7 and 8 of TP53 from cancers of brain, breast, skin, colon, oesophagus, liver, head and neck, stomach and lung (smokers and non-smokers) and spectra induced by benzo[a]pyrene diol epoxide, ultraviolet (UV) B, UVC, simulated sunlight and hydroxyl radicals in the cII, supF and yeast p53 model systems. We demonstrate that this approach allows human cancer and mutagen-induced signatures to be grouped together according to similarity. Specifically, the analysis reveals key differences between smoking- and non-smoking-related lung cancer for TP53 mutations and the mutability of CpG sites between exons in skin cancer.

Curcumin downregulates the inflammatory cytokines CXCL1 and -2 in breast cancer cells via NF{kappa}B

2008-04-01

The dietary antioxidant Curcumin has been proposed for cancer chemoprevention since it induces apoptosis and inhibits the formation of breast cancer metastases. Curcumin acts through the inhibition of phosphorylation of the inhibitor of kappa B (IB), which in turn reduces the nuclear translocation of nuclear factor kappa B (NFB), an inflammation- and cell survival-related transcription factor. However, it is not clear whether the strong antimetastatic effect can exclusively be explained by inhibition of NFB. Here, we addressed the effects of Curcumin (IC₅₀ = 17 µM) in MDA-MB-231 breast cancer cells using microarray gene expression analyses. Among the 62 genes whose expression was significantly altered, we found the two inflammatory cytokines CXCL1 and -2 (Gro and -β) that were downregulated. Further validation of the microarray results by quantitative real-time reverse transcription–polymerase chain reaction, western blots and enzyme-linked immunosorbent assay revealed that Curcumin impairs transcription of CXCL1 and -2 >24 h and reduces the corresponding proteins. Using small interfering RNA techniques, we elucidated the underlying molecular mechanism revealing that reduction of CXCL1 and -2 messenger RNA levels is NFB dependent and requires intact IB expression. Moreover, CXCL1 and -2 silencing leads to downregulation of several metastasis-promoting genes among which we found the cytokine receptor CXCR4. We therefore suggest that the decrease of CXCL1 and -2 mediated by Curcumin is involved in the inhibition of metastasis.

Dietary fish oil and pectin enhance colonocyte apoptosis in part through suppression of PPAR{delta}/PGE2 and elevation of PGE3

2008-04-01

We have shown that dietary fish oil and pectin (FP) protects against radiation-enhanced colon cancer by upregulating apoptosis in colonic mucosa. To investigate the mechanism of action, we provided rats (n = 40) with diets containing the combination of FP or corn oil and cellulose (CC) prior to exposure to 1 Gy, 1 GeV/nucleon Fe-ion. All rats were injected with a colon-specific carcinogen, azoxymethane (AOM; 15 mg/kg), 10 and 17 days after irradiation. Levels of colonocyte apoptosis, prostaglandin E₂ (PGE₂), PGE₃, microsomal prostaglandin E synthase-2 (mPGES-2), total β-catenin, nuclear β-catenin staining (%) and peroxisome proliferator-activated receptor (PPAR) expression were quantified 31 weeks after the last AOM injection. FP induced a higher (P < 0.01) apoptotic index in both treatment groups, which was associated with suppression (P < 0.05) of antiapoptotic mediators in the cyclooxygenase (COX) pathway (mPGES-2 and PGE₂) and the Wnt/β-catenin pathway [total β-catenin and nuclear β-catenin staining (%); P < 0.01] compared with the CC diet. Downregulation of COX and Wnt/β-catenin pathways was associated with a concurrent suppression (P < 0.05) of PPAR levels in FP-fed rats. In addition, colonic mucosa from FP animals contained (P < 0.05) a proapoptotic, eicosapentaenoic acid-derived COX metabolite, PGE₃. These results indicate that FP enhances colonocyte apoptosis in AOM-alone and irradiated AOM rats, in part through the suppression of PPAR and PGE₂ and elevation of PGE₃. These data suggest that the dietary FP combination may be used as a possible countermeasure to colon carcinogenesis, as apoptosis is enhanced even when colonocytes are exposed to radiation and/or an alkylating agent.

Tumor angiogenesis suppression by {alpha}-eleostearic acid, a linolenic acid isomer with a conjugated triene system, via peroxisome proliferator-activated receptor {gamma}

Tsuzuki, T., Kawakami, Y. — 2008-04-01

We have shown previously that -eleostearic acid (ESA), a linolenic acid isomer with a conjugated triene system, suppresses tumor growth in vivo. In our earlier study, blood vessels were observed at the tumor surface in control mice, whereas in ESA-treated mice no such vessels were observed and the inner part of the tumor was discolored. These observations suggested that ESA might suppress cancer cell growth through malnutrition via a suppressive effect on tumor angiogenesis. In the current study, the antiangiogenic effects of ESA were investigated in vivo and in vitro. Tumor cell-induced vessel formation was clearly suppressed in mice orally administered ESA at doses of 50 and 100 mg/kg/day in a dose-dependent manner. ESA also inhibited the formation of capillary-like networks by human umbilical vein endothelial cells (HUVEC) and moderately inhibited HUVEC proliferation and migration in a dose-dependent manner. The mechanism by which ESA inhibited angiogenesis was through suppression of the expression of vascular endothelial growth factor receptors 1 and 2, activation of peroxisome proliferator-activated receptor (PPAR) and induction of apoptosis in HUVEC. We thus demonstrated that, like troglitazone, ESA is a PPAR ligand and that it activates PPAR, induces apoptosis in HUVEC and inhibits angiogenesis. Our findings suggest that ESA has potential use as a therapeutic dietary supplement and medicine for minimizing tumor angiogenesis.

Expression of MRP1 and GSTP1-1 modulate the acute cellular response to treatment with the chemopreventive isothiocyanate, sulforaphane

Sibhatu, M. B., Smitherman, P. K., Townsend, A. J., Morrow, C. S. — 2008-04-01

A major component of the anticarcinogenic activity of the dietary chemopreventive agent sulforaphane (SFN) is attributed to its ability to induce expression of phase II detoxification genes containing the antioxidant response element (ARE) within their promoters. Because SFN is a reactive electrophile––readily forming conjugates with glutathione (GSH)––we asked whether expression of glutathione S-transferase (GST) P1-1 and the GSH conjugate efflux pump, multidrug resistance or resistance-associated protein (MRP) 1, would significantly modify the cellular response to SFN exposure. This was investigated using GST- and MRP1-poor parental MCF7 cells and transgenic derivatives expressing GSTP1-1 and/or MRP1. Compared with parental cells, expression of GSTP1-1 alone enhanced the rate of intracellular accumulation of SFN and its glutathione conjugate, SFN-SG––an effect that was associated with increased ARE-containing reporter gene induction. Expression of MRP1 greatly reduced SFN/SFN-SG accumulation and resulted in significant attenuation of SFN-mediated induction of ARE-containing reporter and endogenous gene expression. Coexpression of GSTP1-1 with MRP1 further reduced the level of induction. Depletion of GSH prior to SFN treatment or the substitution of tert-butylhydroquinone for SFN abolished the effects of MRP1/GSTP1-1 on ARE-containing gene induction—indicating that these effects are GSH dependent. Lastly, analysis of NF-E2-related factor 2 (Nrf2)––a transcription factor operating via binding to the ARE––showed that the increased levels of Nrf2 following SFN treatment were considerably less sustained in MRP1-expressing, especially those coexpressing GSTP1-1, than in MRP1-poor cells. These results suggest that the regulating effects of MRP1 and GSTP1-1 expression on SFN-dependent induction of phase II genes are ultimately mediated by altering nuclear Nrf2 levels.

Lycopene inhibits IGF-I signal transduction and growth in normal prostate epithelial cells by decreasing DHT-modulated IGF-I production in co-cultured reactive stromal cells

Liu, X., Allen, J. D., Arnold, J. T., Blackman, M. R. — 2008-04-01

Prostate stromal and epithelial cell communication is important in prostate functioning and cancer development. Primary human stromal cells from normal prostate stromal cells (PRSC) maintain a smooth muscle phenotype, whereas those from prostate cancer (6S) display reactive and fibroblastic characteristics. Dihydrotestosterone (DHT) stimulates insulin-like growth factor-I (IGF-I) production by 6S but not PSRC cells. Effects of reactive versus normal stroma on normal human prostate epithelial (NPE or PREC) cells are poorly understood. We co-cultured NPE plus 6S or PRSC cells to compare influences of different stromal cells on normal epithelium. Because NPE and PREC cells lose androgen receptor (AR) expression in culture, DHT effects must be modulated by associated stromal cells. When treated with camptothecin (CM), NPE cells, alone and in stromal co-cultures, displayed a dose-dependent increase in DNA fragmentation. NPE/6S co-cultures exhibited reduced CM-induced cell death with exposure to DHT, whereas NPE/PRSC co-cultures exhibited CM-induced cell death regardless of DHT treatment. DHT blocked CM-induced, IGF-I-mediated, NPE death in co-cultured NPE/6S cells without, but not with, added anti-IGF-I and anti-IGF-R antibodies. Lycopene consumption is inversely related to human prostate cancer risk and inhibits IGF-I and androgen signaling in rat prostate cancer. In this study, lycopene, in dietary concentrations, reversed DHT effects of 6S cells on NPE cell death, decreased 6S cell IGF-I production by reducing AR and β-catenin nuclear localization and inhibited IGF-I-stimulated NPE and PREC growth, perhaps by attenuating IGF-I's effects on serine phosphorylation of Akt and GSK3β and tyrosine phosphorylation of GSK3. This study expands the understanding of the preventive mechanisms of lycopene in prostate cancer.

Plasminogen activator inhibitor-1 (Pai-1) blockers suppress intestinal polyp formation in Min mice

2008-04-01

Obesity and hyperlipidemia are known to increase colorectal tumor risk. We noticed that Min mice, featuring a defect in the adenomatous polyposis coli (Apc) gene, develop intestinal polyps along with high serum triglyceride (TG) levels up to 10-fold those observed in wild-type mice. In these mice, messenger RNA (mRNA) expression of lipoprotein lipase, which catalyzes hydrolysis of TG, is downregulated. In the present study, we focused on adipocytokines, especially plasminogen activator inhibitor-1 (Pai-1), which is involved in hyperlipidemic status and may promote intestinal polyp formation in Min mice. Serum Pai-1 levels in the 15-week-old male Min mice were eight times higher than in wild-type mice and hepatic Pai-1 mRNA levels were 11-fold increased. In addition, Pai-1 immunostaining was strong in small intestinal epithelial cells of Min mice. Administration of a PAI-1 inhibitor, SK-216, at 25, 50 and 100 p.p.m. doses in the diet for 9 weeks reduced serum Pai-1 levels and hepatic Pai-1 mRNA levels of Min mice to the wild-type levels. Moreover, SK-216 at 50 and 100 p.p.m. significantly reduced total numbers of intestinal polyps to 64 and 56% of the untreated group value, respectively. Serum TG levels were also decreased by 43% at the dose of 100 p.p.m. Administration of 50 p.p.m. SK-116, another PAI-1 inhibitor, for 9 weeks similarly reduced serum Pai-1 levels and total numbers of intestinal polyps to 70% of the untreated group value. These results indicate that Pai-1 induction associated with hypertriglyceridemia may contribute to intestinal polyp formation with Apc deficiency, and PAI-1 could thus be a novel target for colorectal chemopreventive agents.

Chemopreventive effects of a selective cyclooxygenase-2 inhibitor (etodolac) on chemically induced intraductal papillary carcinoma of the pancreas in hamsters

2008-04-01

The present study was designed to determine whether etodolac, a selective cyclooxygenase-2 inhibitor, prevents chemically induced intraductal papillary carcinoma (IPC) in the main pancreatic duct of hamsters. Hamsters were subjected to cholecystoduodenostomy with dissection of the distal end of the common duct. Four weeks after surgery, the surviving hamsters received subcutaneous injections of N-nitrosobis(2-oxopropyl)amine four times at a dose of 10 mg/kg body wt, every 2 weeks. The animals were divided into three groups according to the simultaneous oral intake of a standard pelleted diet containing etodolac at 0% (group CE, n = 30), 0.01% (group ET, n = 21) and 0.04% (group ET4, n = 25), respectively. Hamsters were killed for pathological examination at 36 weeks after the operation. The incidence of induced pancreatic carcinoma was 93, 81 and 72% in groups CE, ET and ET4, respectively. The pancreatic carcinomas were histologically classified into four types, i.e. tubular, papillary, cyst adenocarcinoma and IPC. The incidence of IPC and the number of IPCs per animal were significantly lower in groups ET4 (36% and 0.48) and ET (48% and 0.62) when compared with group CE (67% and 1.30). The proliferating cell nuclear antigen labeling indices in the non-cancerous epithelial cells of the main pancreatic duct were 2.8 and 6.8% in groups ET4 and ET, respectively, and were significantly lower than that in group CE (10.8%). In conclusion, etodolac inhibited N-nitrosobis(2-oxopropyl)amine-induced IPC in hamsters. Suppression of epithelial cell proliferation of the main pancreatic duct was considered as a possible mechanism of cancer prevention in this hamster model.

Protective versus promotional effects of white tea and caffeine on PhIP-induced tumorigenesis and {beta}-catenin expression in the rat

2008-04-01

A 1 year carcinogenicity bioassay was conducted in rats treated with three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)/high-fat (HF) diet, followed by 2% white tea (wt/vol), 0.05% epigallocatechin-3-gallate (EGCG) or 0.065% caffeine as sole source of fluid intake. Thirty-two percent of the PhIP/HF controls survived to 1 year, compared with 50, 48.7 and 18.2% in groups given white tea, EGCG and caffeine, respectively. After 1 year, PhIP/HF controls had tumors in the colon, skin, small intestine, Zymbal’s gland, salivary gland and pancreas. For all sites combined, excluding the colon, tumor incidence data were as follows: PhIP/HF 69.5%, PhIP/HF + EGCG 48.7%, PhIP/HF + white tea 46.9% and PhIP/HF + caffeine 13.3%. Unexpectedly, a higher incidence of colon tumors was detected in rats post-treated with white tea (69%) and caffeine (73%) compared with the 42% incidence in PhIP/HF controls. In the colon tumors, β-catenin mutations were detected at a higher frequency after caffeine posttreatment, and there was a shift toward more tumors harboring substitutions of Gly34 with correspondingly high protein and messenger RNA expression seen for both β-catenin and c-Myc. c-Myc expression exhibited concordance with tumor promotion, and there was a concomitant increase in cell proliferation versus apoptosis in colonic crypts. A prior report described suppression of PhIP-induced colonic aberrant crypts by the same test agents, but did not incorporate a HF diet. These findings are discussed in the context of epidemiological data which do not support an adverse effect of tea and coffee on colon tumor outcome—indeed, some such studies suggest a protective role for caffeinated beverages.

Biological behavior of CIN lesions is predictable by multiple parameter logistic regression models

2008-04-01

Objectives: Progression and regression of premalignant cervical lesions cannot be predicted using conventional cytomorphological or histomorphological parameters. However, markers such as human papillomavirus (HPV) or makers indicating proliferation, genetic instability and chromosomal aberration may be of predictive value assessing short-term biological behavior of cervical intraepithelial neoplasia. In this paper, we have studied the usage of logistic regression models with Ki-67 labeling index (LI), chromosome index for chromosome 1 (CI#1) and aneusomy for chromosome 1 in cervical smears to predict progressive and regressive behavior of premalignant cervical lesions. Methods: Retrospectively, the intake smears of 42 women showing regression in follow-up and of 31 women showing progression in follow-up were assessed. Results: A multiparameter logistic regression model containing the parameters Ki-67 LI, CI#1 and the fraction of cells with four copies of chromosome 1 per nucleus appeared to be the best predicting model, overall correct classification of 93.2% (area under the receiver operating characteristic curve 0.96 ± 0.02). After cross-validation, the model correctly classified 66 of 73 samples (90.4%). Moreover, the model predicted biological behavior perfectly assessing the smear taken subsequently to the intake smear of 46 women. Conclusion: Although measuring parameters indicating proliferation and chromosome 1 aberration is laborious, this study demonstrates that short-term progressive and regressive behavior is highly predictable using a model combing these parameters. We also showed that in the triage management of high-risk human papillomavirus-positive women with minimally abnormal smears applying a model as such can be useful.

Fez1/Lzts1-deficient mice are more susceptible to N-butyl-N-(4-hydroxybutil) nitrosamine (BBN) carcinogenesis

2008-04-01

FEZ1/LZTS1 is a tumor suppressor gene that is frequently altered in human cancers of different histotypes. We have reported previously that LZTS1 is downregulated in high-grade bladder cancer and that its restoration suppresses tumorigenicity in urothelial carcinoma cells. To further investigate the role of LZTS1 in the development of bladder cancer, we utilized heterozygous and nullizygous Lzts1 mice in a chemically induced carcinogenesis model. Fifty-eight mice consisting of 25 Lzts1^+/+, 17 Lzts1^+/– and 16 Lzts1^–/– were treated with N-butyl-N-(4-hydroxybutil) nitrosamine (BBN). Results showed that there was a significant increase in neoplastic lesions in the Lzts1^+/– (82.3%) and Lzts1^–/– (93.8%) versus Lzts1^+/+ (8.0%) mice after BBN treatment. No difference in cancer incidence between Lzts1^+/– and Lzts1^–/– was observed. Collectively, these findings indicate that loss of one or both LZTS1 alleles hampers the normal defenses of urothelial cells against carcinogens, favoring bladder cancer development. Therefore, LZTS1 may become an excellent target for gene therapy in advanced bladder carcinoma.

BAG-1 is up-regulated in colorectal tumour progression and promotes colorectal tumour cell survival through increased NF-{kappa}B activity

2008-04-01

Although expression of the anti-apoptotic protein Bcl-2-associated athanogene-1 (BAG-1) has been reported as up-regulated in a number of malignancies, we show for the first time that BAG-1 is over-expressed in medium/large-sized colorectal adenomas and carcinomas compared with normal epithelium. To investigate whether expression of BAG-1 is important for colorectal tumour cell survival, microarray analysis was carried out on the HCT116 colorectal carcinoma cell line following transfection with BAG-1 small interfering RNA (siRNA). Analysis identified altered expression of a subset of potential nuclear factor-B (NF-B)-regulated genes. Furthermore, knock down of BAG-1 was shown to inhibit NF-B transcriptional activity. Inhibition of NF-B activity using BAG-1 siRNA or the NF-B inhibitor BAY-117082 suppressed HCT116 cell yield and induced apoptosis; combined treatment had no additive effect, suggesting that the decrease in cell yield associated with knock down of BAG-1 expression is mediated via inhibition of NF-B. Of clinical relevance, BAG-1 siRNA sensitized colorectal carcinoma cells to apoptosis induced by potential therapeutic agent TRAIL as well as tumour necrosis factor-, both inducers of NF-B activity. In summary, knock down of BAG-1 leads to inhibition of NF-B, identifying BAG-1 as a novel regulator of NF-B. It is proposed that, by inhibiting NF-B, suppression of BAG-1 could represent a novel strategy to impede colorectal cancer cell survival and as an adjuvant increase sensitivity to current therapeutic regimes.

SAG/ROC2/RBX2 E3 ligase promotes UVB-induced skin hyperplasia, but not skin tumors, by simultaneously targeting c-Jun/AP-1 and p27

He, H., Gu, Q., Zheng, M., Normolle, D., Sun, Y. — 2008-04-01

Sensitive to apoptosis gene (SAG)/regulator of cullins-2/RING box protein 2 is a stress-responsive RING component of Skp-1/Cullins/F-box protein E3 ubiquitin ligase. When overexpressed, SAG inhibits apoptosis induced by reactive oxygen species or hypoxia. Here, we report that SAG overexpression inhibits ultraviolet (UV) B-induced apoptosis in mouse JB6 epidermal cells. Using a transgenic mouse model, in which SAG expression was targeted primarily to epidermis by a K14 promoter, we showed that, at the early stage of UVB skin carcinogenesis (10 weeks post-UVB exposure), c-Jun, p27, p53, c-Fos and cyclin D1 were strongly induced. While having no effect on UVB-induced p53, c-Fos and cyclin D1, SAG-transgenic expression reduced the levels of c-Jun and p27 and inhibited AP-1 activity. The net outcome of SAG-mediated inhibition of c-Jun/AP-1 (pro-tumor promotion) and of p27 (antiproliferation) increased skin hyperplasia, with no apparent effect on apoptosis, as evidenced by increased skin thickness, and increased rate of DNA synthesis, but hardly any apoptosis. Although skin hyperplasia was promoted, SAG-transgenic expression had no significant effect on tumor formation in the later stage of UVB carcinogenesis. Thus, by simultaneously targeting c-Jun and p27, SAG accelerates UVB-induced skin hyperplasia, but not carcinogenesis.

Mgmt deficiency alters the in vivo mutational spectrum of tissues exposed to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

2008-04-01

It has been proposed that O⁶-methylguanine DNA methyltransferase (MGMT) gene silencing in premalignant lesions and cancers of the lung might result in the acquisition of a ‘mutator’ phenotype. Previously, however, we found that Mgmt^–/– mouse DNA failed to show an increase in spontaneous mutations. We thus hypothesized that only during exposure to specific environmental carcinogens would the consequences of MGMT deficiency become evident. Metabolism of the tobacco-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) generates alkylating species that can react with the O⁶ position of deoxyguanine, thereby yielding substrates for MGMT-mediated repair. To investigate how MGMT might regulate the mutational effects of NNK, Mgmt^–/– mice were crossed with a lacI-based transgenic reporter line (Big Blue^TM) thus enabling an assessment of the in vivo mutagenic effects of this agent. We observed the induction of a complex spectrum of NNK-dependent lacI mutations in both control and Mgmt^–/– tissues, but only a trend in the mutant frequency increases that could be attributed to MGMT deficiency. The mutational spectra of NNK-treated Mgmt^–/– lungs revealed an increase in the absolute number of G:C to A:T changes accompanied by a shift in these from CpG to GpG sites, consistent with an S_N1 alkylation mechanism. In keeping with the high levels of MGMT expressed in the liver, more pronounced mutagenic effects and greater differences in O⁶ position of deoxyguanosine adduct levels following NNK were observed in Mgmt^–/– versus wild-type mice. Extrapolating to humans, MGMT-deficient cells would likely exhibit an increased mutational burden, but only following exposures to specific environmental mutagens such as NNK.

p53 codon 72 polymorphism associated with risk of human papillomavirus-associated squamous cell carcinoma of the oropharynx in never-smokers

2008-04-01

The tumor suppressor p53 protein can be bound, degraded and inactivated by the human papillomavirus (HPV) E6 oncoprotein. The p53 protein's susceptibility to this oncoprotein may be influenced by the p53 codon 72 polymorphism, but the role of such a polymorphism in the development of HPV16-associated squamous cell carcinoma of the oropharynx (SCCOP) has not been established. To investigate the role of the p53 codon 72 polymorphism in the risk of HPV16-associated SCCOP, we conducted a hospital-based case–control study of 188 non-Hispanic white patients with newly diagnosed SCCOP and 342 cancer-free control subjects frequency matched by age (±5 years), sex, tobacco smoking status and alcohol drinking status. We found that HPV16 seropositivity was associated with an increased risk of SCCOP [adjusted odds ratio (OR), 5.7; 95% confidence interval (CI), 3.7–8.7], especially among never-smokers (adjusted OR, 14.1; 95% CI, 6.0–32.9) and among subjects with the p53 codon 72 variant genotypes [Arginine (Arg)/Proline (Pro) and Pro/Pro] (adjusted OR, 9.2; 95% CI, 4.7–17.7). A significant multiplicative interaction on the risk of SCCOP was also found between the p53 codon 72 polymorphism and HPV16 seropositivity (P = 0.05). Among never-smokers, the risk of SCCOP for those who had both HPV16 seropositivity and p53 codon 72 variant genotypes (Arg/Pro + Pro/Pro) was particularly high (adjusted OR, 22.5; 95% CI, 4.8–106.2). These findings suggest that p53 codon 72 variant genotypes modify the risk of HPV16-associated SCCOP and may be markers of genetic susceptibility to HPV16-associated SCCOP, especially among never-smokers.

A protective role of mast cells in intestinal tumorigenesis

2008-04-01

Mast cells have been observed in numerous types of tumors; however, their role in carcinogenesis remains poorly understood. The majority of epidemiological evidence suggests a negative association between the presence of mast cells and tumor progression in breast, lung and colonic neoplasms. Intestinal adenomas in the multiple intestinal neoplasia (Min, APC^Min/+) mouse displayed increased numbers of mast cells and increased abundance of mast cell-associated proteinases as determined by transcriptional profiling with the Hu/Mu ProtIn microarray. To examine the role of mast cells in intestinal tumorigenesis, a mutant mouse line deficient in mast cells, Sash mice (c-kit^W-sh/W-sh), was crossed with the Min mouse, a genetic model of intestinal neoplasia. The resulting mast cell-deficient Min–Sash mice developed 50% more adenomas than littermate controls and the tumors were 33% larger in Min–Sash mice. Mast cell deficiency did not affect tumor cell proliferation; however, apoptosis was significantly inhibited in mast cell-deficient mice. Mast cells have been shown to act as critical upstream regulators of numerous inflammatory cells. Neutrophil, macrophage and T cell populations were similar between Min and Min–Sash mice; however, eosinophils were significantly less abundant in tumors obtained from Min–Sash animals. These results indicate a protective, antitumor role of mast cells in a genetic model of early-stage intestinal tumorigenesis.