Carcinogenesis - recent issues

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Mon, 05 Oct 2009 17:18:02 PDT

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Mon, 05 Oct 2009 17:18:02 PDT

Involvement of AdipoR receptor in adiponectin-induced motility and {alpha}2{beta}1 integrin upregulation in human chondrosarcoma cells

Mon, 05 Oct 2009 17:18:02 PDT

Chondrosarcoma is a type of highly malignant tumor with a capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and is involved in energy homeostasis. However, the effect of adiponectin on migration activity in human chondrosarcoma cells is mostly unknown. We found that adiponectin increased the migration and expression of 2β1 integrin in human chondrosarcoma cells. The protein and messenger RNA expression of adiponectin receptor (AdipoR1 and AdipoR2) in chondrosarcoma patients and chondrosarcoma cell lines were significantly higher than the normal cartilage. Moreover, primary chondrosarcoma and chondrosarcoma cell lines (SW1353 and JJ012) were more invasive than normal chondrocytes. Adiponectin-mediated migration and integrin expression was attenuated by 5'-adenosine monophosphate-activated protein kinase (AMPK) small interfering RNA and an AMPK inhibitor (Ara A and compound C). Activation of p38 and nuclear factor-kappa B (NF-B) pathways after adiponectin treatment was demonstrated, and adiponectin-induced expression of integrins and migration activity was inhibited by the specific inhibitor and mutant of p38 and NF-B cascades. This study showed for the first time that adiponectin mediates the migration of human chondrosarcoma cells. One mechanism underlying adiponectin-directed migration was transcriptional upregulation of 2β1 integrin and activation of AdipoR receptor, AMPK, p38 and NF-B pathways.

Secreted LOXL2 is a novel therapeutic target that promotes gastric cancer metastasis via the Src/FAK pathway

Mon, 05 Oct 2009 17:18:02 PDT

The purpose of this study was to investigate invasion- and metastasis-related genes in gastric cancer. To this end, we used the transwell system to select a highly invasive subcell line from minimally invasive parent cells and compared gene expression in paired cell lines with high- and low-invasive potentials. Lysyl oxidase-like 2 (LOXL2) was overexpressed in the highly invasive subcell line. Immunohistochemical analysis revealed that LOXL2 expression was markedly increased in carcinoma relative to normal epithelia, and this overexpression in primary tumor was significantly associated with depth of tumor invasion, lymph node metastasis and poorer overall survival. Moreover, LOXL2 expression was further increased in lymph node metastases compared with primary cancer tissues. RNA interference-mediated knockdown and ectopic expression of LOXL2 showed that LOXL2 promoted tumor cell invasion in vitro and increased gastric carcinoma metastasis in vivo. Subsequent mechanistic studies showed that LOXL2 could activate both the Snail/E-cadherin and Src kinase/Focal adhesion kinase (Src/FAK) pathways. However, secreted LOXL2 induced gastric tumor cell invasion and metastasis exclusively via the Src/FAK pathway. Expression correlation analysis in gastric carcinoma tissues also revealed that LOXL2 promoted invasion via the Src/FAK pathway but not the Snail/E-cadherin pathway. We then evaluated secreted LOXL2 as a target for gastric carcinoma treatment and found that an antibody against LOXL2 significantly inhibited tumor growth and metastasis. Overall, our data revealed that LOXL2 overexpression, a frequent event in gastric carcinoma progression, contributes to tumor cell invasion and metastasis, and LOXL2 may be a therapeutic target for preventing and treating metastases.

Oncogenic Ras, but not V600EB-RAF, protects from cholesterol depletion-induced apoptosis through the PI3K/AKT pathway in colorectal cancer cells

Calleros, L., Sanchez-Hernandez, I., Baquero, P., Toro, M. J., Chiloeches, A. — Mon, 05 Oct 2009 17:18:02 PDT

Cholesterol is necessary for proliferation and survival of transformed cells. Here we analyse the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in colorectal cancer cells carrying oncogenic Ras or ^V600EB-RAF mutations. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment results in a significant increase in apoptosis in HT-29 and Colo-205 cells containing the ^V600EB-RAF mutation, but not in HCT-116 and LoVo cells harbouring the ^G13DRas mutation, or BE cells, which possess two mutations, ^G13DRas and ^G463VB-RAF. We also demonstrate that oncogenic Ras protects from apoptosis induced by cholesterol depletion through constitutive activation of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. The specific activation of the PI3K/AKT pathway by overexpression of the ^V12RasC40 mutant or a constitutively active AKT decreases the LPDS plus 25-HC-induced apoptosis in HT-29 cells, whereas PI3K inhibition or abrogation of AKT expression renders HCT-116 sensitive to cholesterol depletion-induced apoptosis. Moreover, our data show that LPDS plus 25-HC increases the activity of c-Jun N-terminal kinase proteins only in HT-29 cells and that the inhibition of this kinase blocks the apoptosis induced by LPDS plus 25-HC. Finally, we demonstrate that AKT hyperactivation by oncogenic Ras protects from apoptosis, preventing the activation of c-Jun N-terminal kinase by cholesterol depletion. Thus, our data demonstrate that low levels of cholesterol induce apoptosis in colorectal cancer cells without oncogenic Ras mutations. These results reveal a novel molecular characteristic of colon tumours containing Ras or B-RAF mutations and should help in defining new targets for cancer therapy.

The human homolog of the Drosophila headcase protein slows down cell division of head and neck cancer cells

Dowejko, A., Bauer, R. J., Muller-Richter, U. D.A., Reichert, T. E. — Mon, 05 Oct 2009 17:18:02 PDT

The human homolog of the Drosophila headcase (HECA) belongs to a new class of cell differentiation regulators. In Drosophila, the HECA protein regulates the proliferation and differentiation of cells during adult morphogenesis. There is growing evidence that HECA plays an important role in human carcinogenesis. In different tumor entities, an altered HECA expression was found (colorectal, pancreatic and renal cancer). Colorectal cancer studies also suggested HECA as a marker for early disease stages. Therefore, we speculated whether human HECA affects cell cycle progression and proliferation in head and neck cancer cells. In vivo, we found a distinct HECA protein expression in basal and superficial cells of a healthy oral epithelium via immunohistochemistry, whereas in tissues of oral squamous cell carcinoma (OSCC), a weaker staining was observed, particularly in basal cells. In vitro, mRNA and protein expression analyses of OSCC cell lines exhibited that HECA expression correlates with the state of cellular differentiation. In further investigations, we overexpressed HECA in the OSCC cell line PCI 13 and performed functional assays. HECA-overexpressing OSCC cells revealed a significant extended doubling time (up to 45%, 17 h) and yielded a lower number of proliferating cells (up to 30%) than controls. Flow cytometry analyses have shown that HECA-overexpressing OSCC cells forced to hold in the G₂/M-Phase. In summary, our results show that human HECA slows down cell division of OSCC cells and may therefore act as a tumor suppressor in head and neck cancer.

Intercellular communication of cellular stress monitored by {gamma}-H2AX induction

Mon, 05 Oct 2009 17:18:02 PDT

When cells are exposed to ionizing radiation (IR), unexposed cells that share media with damaged cells exhibit similar effects to irradiated cells including increased levels of DNA double-strand breaks (DSBs). Hypothesizing that this effect, known as the radiation-induced bystander effect, may be a specific instance of communication between damaged and undamaged cells regardless of damage source, we demonstrated that exposure of target cells to non-IR induces bystander damage in non-targeted cells as measured by -H2AX and 53BP1 focal formation. Initially, bystander damage was found primarily in S-phase cells, but at later times, non-S-phase cells were also affected. In addition, media from undamaged malignant and senescent cells also was found to induce DSBs in primary cultures. Media conditioned on cells targeted with either ionizing or non-IR as well as on undamaged malignant and senescent cells contained elevated levels of several cytokines. One of these, transforming growth factor beta (TGF-β), and nitric oxide (NO) were found to elevate numbers of -H2AX/53BP1 foci in normal cell cultures similar to levels found in bystander cells, and this elevation was abrogated by NO synthase inhibitors, TGF-β blocking antibody and antioxidants. These findings support the hypothesis that damage in bystander cells results from their exposure to cytokines or reactive compounds released from stressed cells, regardless of damage source. These results have implications for oncogenesis in that they indicate that damaged normal cells or undamaged tumor cells may induce genomic instability, leading to an increased risk of oncogenic transformation in other cells with which they share media or contact directly.

CRK7 modifies the MAPK pathway and influences the response to endocrine therapy

Iorns, E., Martens-de Kemp, S. R., Lord, C. J., Ashworth, A. — Mon, 05 Oct 2009 17:18:03 PDT

Endocrine therapies, which inhibit estrogen receptor (ER) signaling, are the most common and effective treatment for ER-positive breast cancer. However, the use of these agents is limited by the frequent development of resistance. The cyclin-dependent kinase family member CRK7 (aka CRKRS) was identified from an RNA interference screen for modifiers of tamoxifen sensitivity. Here, we demonstrate that silencing of CRK7 not only causes resistance to tamoxifen but also leads to resistance to additional endocrine therapies including ICI 182780 and estrogen deprivation, a model of aromatase inhibition. We show that CRK7 silencing activates the mitogen-activated protein kinase (MAPK)-signaling pathway, which causes a loss of ER dependence, resulting in endocrine therapy resistance. This study identifies a novel role for CRK7 in MAPK regulation and resistance to estrogen signaling inhibitors.

OSU-A9, a potent indole-3-carbinol derivative, suppresses breast tumor growth by targeting the Akt-NF-{kappa}B pathway and stress response signaling

Mon, 05 Oct 2009 17:18:03 PDT

The molecular heterogeneity of human tumors challenges the development of effective preventive and therapeutic strategies. To overcome this issue, a rational approach is the concomitant targeting of clinically relevant cellular abnormalities with combination therapy or a potent multi-targeted agent. OSU-A9 is a novel indole-3-carbinol derivative that retains the parent compound's ability to perturb multiple components of oncogenic signaling, but provides marked advantages in chemical stability and antitumor potency. Here, we show that OSU-A9 exhibits two orders of magnitude greater potency than indole-3-carbinol in inducing apoptosis in various breast cancer cell lines with distinct genetic abnormalities, including MCF-7, MDA-MB-231 and SKBR3, with the half maximal inhibitory concentration in the range of 1.2–1.8 µM vis-à-vis 200 µM for indole-3-carbinol. This differential potency was paralleled by OSU-A9’s superior activity against multiple components of the Akt–nuclear factor-kappa B (NF-B) and stress response signaling pathways. Notable among these were the increased estrogen receptor (ER)-β/ER expression ratio, reduced expression of HER2 and CXCR4 and the upregulation of aryl hydrocarbon receptor expression and its downstream target NF-E2 p45-regulated factor (Nrf2). Non-malignant MCF-10A cells were resistant to OSU-A9’s antiproliferative effects. Daily oral administration of OSU-A9 at 25 and 50 mg/kg for 49 days significantly inhibited MCF-7 tumor growth by 59 and 70%, respectively, without overt signs of toxicity or evidence of induced hepatic biotransformation enzymes. In summary, OSU-A9 is a potent, orally bioavailable inhibitor of the Akt–NF-B signaling network, targeting multiple aspects of breast tumor pathogenesis and progression. Thus, its translational potential for the treatment or prevention of breast cancer warrants further investigation.

Involvement of p29 in DNA damage responses and Fanconi anemia pathway

Chu, P.-C., Wang, T.-Y., Lu, Y.-T., Chou, C.-K., Yang, Y.-C., Chang, M.-S. — Mon, 05 Oct 2009 17:18:03 PDT

Human p29 is a chromatin-associated protein and the silencing of p29 expression increases cell population in G₁ phase and decreases phosphorylation levels of Chk1 and Chk2 in response to UV treatment. To further characterize the function of p29, U2OS and Fanconi anemia complementation group G (FA-G) cells with constitutive p29 expression have been established. Analyses of these cells identified increased phosphorylation levels of Chk1 and Chk2, which were accompanied by elevated amounts of chromatin-associated Mre11–Rad50–Nbs1 complex and ATR-IP. Monoubiquitination of the FA ID complex was restored in p29 stably expressing FA-G cells. Moreover, lower tumor incidence was observed in mp29 transgenic mice after UV irradiation. These results suggest the involvement of p29 in the DNA damage responses and Fanconi anemia pathway.

A novel functional variant (-842G>C) in the PIN1 promoter contributes to decreased risk of squamous cell carcinoma of the head and neck by diminishing the promoter activity

Mon, 05 Oct 2009 17:18:03 PDT

PIN1, a new peptidyl-prolyl cis/trans isomerase, regulates the conformation of Pro-directed phosphorylation sites, revealing a new postphosphorylation regulatory mechanism. PIN1-induced conformational changes potentiate multiple oncogenic signaling pathways, and PIN1 overexpression is reported as a prevalent and specific event in human cancers. In this study, we tested the hypothesis that common polymorphisms in the coding and promoter regions of PIN1 are associated with risk of squamous cell carcinoma of the head and neck (SCCHN). We genotyped three selected PIN1 polymorphisms (–842G>C, –667T>C and Gln33Gln) in a hospital-based case–control study of 1006 patients with SCCHN and 1007 cancer-free control subjects. We found that the –842C variant genotypes were associated with decreased risk for SCCHN [Odds Ratio (OR) = 0.74; 95% confidence interval (CI) = 0.59–0.93 for the CG genotype, OR = 0.82; 95% CI = 0.34–2.01 for the CC genotype and OR = 0.74; 95% CI = 0.59–0.93 for CG+CC genotypes, compared with the GG genotype]. However, no altered risks were observed for –667T>C and Gln33Gln polymorphisms. Further experiments of the reporter gene expression driven by the allelic PIN1 promoter showed that the –842G allele had a higher activity than that driven by the –842C allele, suggesting that the –842C allele was associated with a reduced transcriptional activity, a finding consistent with a reduced risk observed in the case–control analysis. Large prospective studies of diverse ethnic groups and diverse cancer sites are warranted to validate our findings.

Human papillomavirus type 16 and 18 in primary lung cancers--a meta-analysis

Srinivasan, M., Taioli, E., C.Ragin, C. — Mon, 05 Oct 2009 17:18:03 PDT

Lung cancer is the leading cause of cancer mortality worldwide. A possible carcinogenic role of human papillomavirus (HPV) has been investigated for >20 years and has major clinical and public health implications. We performed a meta-analysis to assess the prevalence of HPV16 and HPV18 in primary lung cancers (2435 subjects from 37 published studies). The overall HPV prevalence ranged from 0.0 to 78.3% with large heterogeneity across geographic regions and histological tissue types. A higher proportion, 50% (7/14), of the European studies reported low or no HPV prevalence (0–10%) compared with the Asian studies, 22% (4/18). When the analysis was limited to HPV16 and HPV18 prevalence, a higher prevalence in Asia (HPV16 = 11.6% and HPV18 = 8.8%) than in Europe (HPV16 = 3.5% and HPV18 = 3.6%) was observed. Studies using HPV-specific primers resulted in higher prevalence rates than consensus HPV primers (HPV16: Asia = 13% and Europe = 6%; HPV18: Asia = 13% and Europe = 5%). Further studies are needed to elucidate the role of HPV in lung carcinogenesis with careful thought given to study design and laboratory detection methods for a more accurate assessment of HPV status in lung tumors.

Interaction between cytochrome P450 1A2 genetic polymorphism and cigarette smoking on the risk of hepatocellular carcinoma in a Japanese population

Mon, 05 Oct 2009 17:18:03 PDT

Limited epidemiological evidence suggests that genetic polymorphisms of drug-metabolizing enzymes such as cytochrome P450 (CYP), glutathione S-transferase (GST) and N-acetyltransferase (NAT) may be involved in tobacco-related hepatocarcinogenesis. We conducted a case–control study, including 209 incident cases with hepatocellular carcinoma (HCC) and two different control groups [275 hospital controls and 381 patients with chronic liver disease (CLD) without HCC], to investigate whether CYP1A1, CYP1A2, CYP2A6, CYP2E1, GSTM1 and NAT2 polymorphisms are related to the risk of HCC with any interaction with cigarette smoking. Overall, no significant associations with HCC were observed for any genotypes against either control group. However, we found a significant interaction (P = 0.0045) between CYP1A2 -3860G>A polymorphism and current smoking on HCC risk when we compared HCC cases with CLD patients; adjusted odds ratios [ORs; and 95% confidence intervals (CIs)] for G/A and A/A genotypes relative to G/G genotype were 0.28 (0.12–0.66) and 0.18 (0.04–0.94), respectively, among current smokers (P trend = 0.002), as compared with 1.28 (0.80–2.06) and 0.76 (0.34–1.71), respectively, among never/former smokers (P trend = 0.96). Similarly, in CYP1A2 G/G genotype, significant risk increase was observed for current smoking (OR = 4.08, 95% CI = 2.02–8.25) or more recent cigarette use (e.g. pack-years during last 5 years, P trend = 0.0003) but not in G/A and A/A genotypes combined (OR for current smoking = 1.39, 95% CI = 0.63–3.03; P trend for pack-years during last 5 years = 0.40). These results suggest that the CYP1A2 -3860G>A polymorphism modifies the smoking-related HCC risk among CLD patients.

DNA repair gene polymorphisms and risk of cutaneous melanoma: a systematic review and meta-analysis

Mocellin, S., Verdi, D., Nitti, D. — Mon, 05 Oct 2009 17:18:03 PDT

Polymorphisms of DNA repair-related genes might modulate cancer predisposition. We performed a systematic review and meta-analysis of the available evidence regarding the relationship between these polymorphisms and the risk of developing cutaneous melanoma. Relevant studies were searched using PubMed, Medline, Embase, Cancerlit, Cochrane and ISI Web of Knowledge databases. Data were gathered according to the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines. The model-free approach was adopted to perform the meta-analysis of the retrieved data. We identified 20 original reports that describe the relationship between melanoma risk and the single-nucleotide polymorphisms (SNPs) of 16 genes (cases = 4195). For seven SNPs considered in at least two studies, the findings were heterogeneous. Data were suitable for meta-analysis only in the case of the XPD/ERCC2 SNP rs13181 (cases = 2308, controls = 3698) and demonstrated that the variant C allele is associated with increased melanoma risk (odds ratio = 1.12, 95% confidence interval = 1.03–1.21, P = 0.01; population attributable risk = 9.6%). This is the first meta-analysis suggesting that XPD/ERCC2 might represent a low-penetrance melanoma susceptibility gene. Much work is still to be done before definitive conclusions can be drawn on the role of DNA repair alterations in melanomagenesis since for the other genes involved in this highly complex process, the available information is scarce or null.

Benzyl isothiocyanate-mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of MAPK in human pancreatic cancer cells

Sahu, R. P., Zhang, R., Batra, S., Shi, Y., Srivastava, S. K. — Mon, 05 Oct 2009 17:18:03 PDT

In our previous studies, we have shown that benzyl isothiocyanate (BITC) inhibits the growth of human pancreatic cancer cells by inducing apoptosis. In the present study, we demonstrate the activation of all the three (MAPK) family members [extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK) and P38] in response to BITC treatment. Exposure of Capan-2 cells with varying concentrations of BITC for 24 h resulted in the phosphorylation (activation) of ERK at Thr202/Tyr204, JNK at Thr183/Tyr185 and P38 at Thr180/Tyr182, leading to the induction of apoptosis. Similar MAPK activation was also observed in MiaPaCa-2 cells in response to BITC treatment. However, normal human pancreatic ductal epithelial cells did not show the activation of MAPK's and remained unaffected by BITC treatment. To confirm the role of ERK, JNK and P38 in BITC-induced G₂/M arrest and apoptosis, Capan-2 cells were pre-treated with MAPK-specific inhibitors or MAPK8-short hairpin RNA (shRNA) prior to BITC treatment. Significant protection from BITC-induced G₂/M arrest was observed in the cells pre-treated with MAPK kinase (MEK-1) but not JNK or P38 inhibitors. On the other hand, BITC-induced apoptosis was almost completely abrogated in the cells pre-treated with MEK-1, JNK or P38 inhibitors. Similarly, MAPK8-shRNA also offered almost complete protection against BITC-induced G₂/M arrest and apoptosis. Furthermore, we observed that BITC treatment leads to the generation of reactive oxygen species (ROS) in Capan-2 and MiaPaCa-2 cells, which in part was orchestrated by depletion of reduced glutathione (GSH) level. Blocking ROS generation with N-acetyl-L-cysteine (NAC) significantly prevented GSH depletion and activation of ERK and JNK but not P38. Further, NAC or tiron prevented G₂/M arrest by blocking G₂/M regulatory proteins and completely protected the cells from BITC-induced apoptosis. Taken together, our results suggest that BITC-mediated G₂/M arrest is mediated through ERK activation, whereas apoptosis is via ERK, JNK and P38.

1-Cyano-2,3-epithiopropane is a novel plant-derived chemopreventive agent which induces cytoprotective genes that afford resistance against the genotoxic {alpha},{beta}-unsaturated aldehyde acrolein

Mon, 05 Oct 2009 17:18:03 PDT

Epithionitriles represent a previously unrecognized class of cancer chemopreventive phytochemical generated from alkenyl glucosinolates in cruciferous vegetables. In rat liver RL-34 epithelial cells, 1-cyano-2,3-epithiopropane (CETP), 1-cyano-3,4-epithiobutane (CETB) and 1-cyano-4,5-epithiopentane (CETPent) were shown to induce cytoprotective enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione (GSH) S-transferase A3 and the glutamate–cysteine ligase modifier subunit; CETP was more potent in this regard than were either CETB or CETPent, with 50 µM CETP eliciting a remarkable ~10-fold induction of NQO1. Furthermore, 50 µM CETP stimulated a 2.0-fold overproduction of GSH in RL-34 cells. Transfection experiments demonstrated that epithionitriles induced gene expression through an antioxidant response element (ARE) and that transactivation of an Nqo1-luciferase reporter plasmid was dependent on NF-E2 p45-related factor 2 (Nrf2), a cap'n'collar basic region leucine zipper transcription factor. Evidence is presented that CETP affected Nrf2-mediated induction of ARE-driven transcription by inhibiting Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase substrate adaptor that negatively regulates Nrf2. We found that Nqo1 was expressed constitutively at high levels in Keap1^–/– mouse embryonic fibroblasts (MEFs) and it was not further induced by CETP. However, knock-in of mouse Keap1 or zebrafish Keap1a into Keap1^–/– MEFs repressed Nqo1-luciferase reporter gene activity, but repression by the murine or zebrafish proteins was antagonized by CETP. Pre-treatment of Nrf2^+/+ MEFs, but not Nrf2^–/– MEFs, with 15 µM CETP for 24 h conferred 2.4-fold resistance against subsequent exposure to the ,β-unsaturated aldehyde acrolein, indicating that the phytochemical exerts chemopreventive properties against genotoxic xenobiotics.

Chemopreventative effect of an inducible nitric oxide synthase inhibitor, ONO-1714, on inflammation-associated biliary carcinogenesis in hamsters

Mon, 05 Oct 2009 17:18:03 PDT

The present study was designed to investigate whether an inducible nitric oxide synthase (iNOS)-specific inhibitor, ONO-1714 [(1S, 5S, 6R, 7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0] heptane], could prevent inflammation-associated biliary carcinogenesis in bilioenterostomized hamsters. Syrian golden hamsters underwent choledochojejunostomy and then received subcutaneous injections of the chemical carcinogen N-nitrosobis(2-oxopropyl)amine every 2 weeks at a dose of 10 mg/kg body wt, starting 4 weeks after surgery and continuing for 18 weeks. The hamsters were divided into two groups according to their oral intake of either a standard pelleted diet containing ONO-1714 at 100 p.p.m. for 18 weeks (ONO group, n = 15) or an ordinary diet alone (control group, n = 15). The animals were killed 22 weeks after surgery, and the development of biliary tumors was examined histologically. The presence and degree of cholangitis, cell kinetic status of the biliary epithelium and iNOS expression were evaluated. Intrahepatic biliary adenomas developed in all control animals, whereas they developed in only seven (47%) hamsters treated with ONO-1714 (P < 0.05). Intrahepatic biliary carcinomas were present in 13 (87%) hamsters in the control group and in only 6 (40%) hamsters in the ONO groups (P < 0.05). Histological and immunohistochemical examinations demonstrated a significant decrease in the degree of cholangitis, biliary epithelial cell kinetics and the expression of iNOS in the biliary epithelium in the ONO group in comparison with the control (P < 0.05). These results indicate that ONO-1714 represses N-nitrosobis(2-oxopropyl)amine-induced biliary carcinogenesis in bilioenterostomized hamsters and inhibits iNOS expression in the biliary epithelium. ONO-1714 may therefore be a promising agent for the prevention of biliary carcinoma in various inflammation-associated biliary disorders.

Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1{alpha}

Mon, 05 Oct 2009 17:18:03 PDT

The interplay among hypoxia-inducible factor 1-alpha (HIF-1), p53 and human orthologue of murine double minute 2 (Hdm2) has been introduced as a key event in tumor promotion and angiogenesis. Recently, nutlin-3, a small-molecule antagonist of Hdm2, was demonstrated to inhibit the HIF-1-mediated vascular endothelial growth factor production and tumor angiogenesis. Yet, the mechanism by which nutlin-3 inhibits HIF-1 is an open question. We here addressed the mode-of-action of nutlin-3 with respect to the HIF-1–p53–Hdm2 interplay. The effect of nutlin-3 on HIF-1 function was examined by reporter analyses, immunoprecipitation and immunoblotting. Nutlin-3 downregulated HIF-1, which occurred p53-dependently but von Hippel-Lindau-independently. On the contrary, nutlin-3 blunted the hypoxic induction of vascular endothelial growth factor by inactivating HIF-1 even in p53-null cells. The C-terminal transactivation domain (CAD) of HIF-1 was inactivated by nutlin-3, and furthermore, the factor-inhibiting hypoxia-inducible factor (FIH) hydroxylation of Asn803 was required for the nutlin-3 action. In terms of protein interactions, Hdm2 competed with FIH in CAD binding and inhibited the Asn803 hydroxylation both in vivo and in vitro, which facilitated p300 recruitment. Moreover, nutlin-3 reinforced the FIH binding and Ans803 hydroxylation by inhibiting Hdm2. In conclusion, Hdm2 functionally activates HIF-1 by inhibiting the FIH interaction with CAD, and the Hdm2 inhibition by nutlin-3 results in HIF-1 inactivation and vascular endothelial growth factor suppression. The interplays among HIF-1, Hdm2, FIH and p300 could be potential targets for treating tumors overexpressing HIF-1.

Increased skin carcinogenesis in caspase-activated DNase knockout mice

Mon, 05 Oct 2009 17:18:03 PDT

Caspase-activated DNase (CAD), also called DNA fragmentation factor (DFF), is the enzyme responsible for DNA fragmentation during apoptosis, a hallmark of programmed cell death. CAD/DFF has been shown to suppress radiation-induced carcinogenesis by preventing genomic instability in cells. In this study, we have investigated the role of CAD in chemical carcinogenesis using CAD-null mice and two-stage model of skin carcinogenesis. After topical treatment of mouse skin with dimethylbenz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoting agent, there was a 4-fold increase in the number of papillomas per mouse and 50.8% increase in the incidence of papilloma formation in the CAD knockout mice compared with wild-type littermates. The papillomas in CAD-null mice grew faster and reached larger sizes. These data indicate that loss of CAD function enhances tumorigenesis induced by a chemical carcinogen in the DMBA/TPA two-stage model of skin carcinogenesis in mice.

P-cadherin induces an epithelial-like phenotype in oral squamous cell carcinoma by GSK-3beta-mediated Snail phosphorylation

Bauer, K., Dowejko, A., Bosserhoff, A.-K., Reichert, T.E., Josef Bauer, R. — Mon, 05 Oct 2009 17:18:03 PDT

Cadherins belong to a family of Ca²⁺-dependent homophilic cell–cell adhesion proteins that are important for correct cellular localization and tissue integrity. They play a major role in the development and homeostasis of epithelial architecture. Recently, it has become more and more evident that P-cadherin contributes to the oncogenesis of many tumors. To analyze the role of P-cadherin in oral squamous cell carcinoma (OSCC), we used a cell line that was deficient of the classical cadherins, P-cadherin, E-cadherin and N-cadherin. This cell line was transfected with full-length P-cadherin (PCI52_PC). After overexpression of P-cadherin, PCI52_PC gained an epithelial-like brickstone morphology in contrast to the mock-transfected cells with a spindle-shaped mesenchymal morphology. Immunohistochemical analysis revealed a strong nuclear Snail staining in mock-transfected cells compared with a significantly reduced nuclear staining and translocation to the cytoplasm in P-cadherin-overexpressing cells. Interestingly, the effects triggered by P-cadherin overexpression could be reversed by transfecting the cells with an antisense P-cadherin plasmid construct. Additional investigations showed a reexpression of E-cadherin in all P-cadherin-transfected cell clones in contrast to the mock controls. Analyzing the signaling mechanism behind it, we found glycogen-synthase-kinase-3beta (GSK-3beta) bound to Snail in all cell clones. Furthermore, P-cadherin-overexpressing cell lines showed activated GSK-3beta that phosphorylated Snail leading to its cytoplasmic translocation. In summary, our results reveal P-cadherin as one major component in reconfiguring mesenchymal cells with epithelial features by triggering GSK-3beta-mediated inactivation and cytoplasmatic translocation of Snail in OSCC.

Targeted mutation of p53 and Rb in mesenchymal cells of the limb bud produces sarcomas in mice

Lin, P. P., Pandey, M. K., Jin, F., Raymond, A.K., Akiyama, H., Lozano, G. — Mon, 05 Oct 2009 17:18:03 PDT

Mice bearing germ line mutations of p53 develop sarcomas at a significant rate. Since they are susceptible to a variety of other malignancies, they are not ideally suited to the study of sarcomas. To test the possibility that targeted mutation of tumor suppressor genes in early mesenchymal cells would induce formation of sarcomas, the Prx1-cre transgenic mouse was crossed to mice-bearing floxed alleles of p53 and Rb. Mice with homozygous deletion of p53 (Prx1-cre p53^lox/lox) developed sarcomas in the extremities at a mean time of 50 weeks. Osteosarcomas (OS) were the most common type of sarcoma (61%) followed by poorly differentiated soft tissue sarcomas (PDSTS) (32%). Homozygous deletion of p53 produced sarcomas significantly more rapidly than heterozygous deletion, which resulted in sarcoma formation after a mean of 96 weeks. Mice with homozygous Rb mutation (Prx1-cre Rb^lox/lox) developed normally and had no ostensible defects in the limbs. In contrast to p53, targeted deletion of Rb did not produce sarcomas in the limbs. However, simultaneous deletion of Rb and p53 accelerated the time to sarcoma formation, and a greater percentage of PDSTS were found. Deletion of p53 in committed osteoblasts by the Col1a1-cre transgenic mouse bearing an osteoblast-specific enhancer resulted in a high percentage of OS. These findings suggest that deletion of p53 in mesenchymal cells that give rise to osteoblasts is a powerful initiator of OS. Deletion of Rb does not initiate sarcoma formation in mice, but it accelerates formation of both soft tissue sarcomas and OS.

HGF/Met signalling promotes PGE2 biogenesis via regulation of COX-2 and 15-PGDH expression in colorectal cancer cells

Mon, 05 Oct 2009 17:18:03 PDT

Evidence points towards a pivotal role for cyclooxygenase (COX)-2 in promoting colorectal tumorigenesis through increasing prostaglandin E₂ (PGE₂) levels. PGE₂ signalling is closely associated with the survival, proliferation and invasion of colorectal cancer cells. Recently, a reduction in PGE₂ inactivation, a process mediated by the nicotinamide adenine dinucleotide (NAD+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), has also been shown to promote tumoral PGE₂ accumulation. The hepatocyte growth factor (HGF) receptor, Met, is frequently over-expressed in colorectal tumours and promotes cancer growth, metastasis and resistance to therapy, although the mechanisms for this have not been fully elucidated. Here, we report that HGF/Met signalling can promote PGE₂ biogenesis in colorectal cancer cells via COX-2 up-regulation and 15-PGDH down-regulation at the protein and messenger RNA level. Pharmacological inhibition of MEK and PI3K suggested that both extracellular signal-regulated kinase (ERK) and AKT signalling are required for COX-2 protein up-regulation and 15-PGDH down-regulation downstream of Met. Notably, inhibition of Met with the small molecule inhibitor SU11274 reduced COX-2 expression and increased 15-PGDH expression in high Met-expressing cells. We also show that hypoxia potentiated HGF-driven COX-2 expression and enhanced PGE₂ release. Furthermore, inhibition of COX-2 impeded the growth-promoting effects of HGF, suggesting that the COX-2/PGE₂ pathway is an important mediator of HGF/Met signalling. These data reveal a critical role for HGF/Met signalling in promoting PGE₂ biogenesis in colorectal cancer cells. Targeting the crosstalk between these two important pathways may be useful for therapeutic treatment of colorectal cancer.

Finding transcriptomics biomarkers for in vivo identification of (non-)genotoxic carcinogens using wild-type and Xpa/p53 mutant mouse models

Mon, 05 Oct 2009 17:18:03 PDT

The carcinogenic potential of chemicals and pharmaceuticals is traditionally tested in the chronic, 2 year rodent bioassay. This assay is not only time consuming, expensive and often with a limited sensitivity and specificity but it also causes major distress to the experimental animals. A major improvement in carcinogenicity testing, especially regarding reduction and refinement of animal experimentation, could be the application of toxicogenomics. The ultimate aim of this study is to demonstrate a proof-of-principle for transcriptomics biomarkers in various tissues for identification of (subclasses of) carcinogenic compounds after short-term in vivo exposure studies. Both wild-type and DNA repair-deficient Xpa^–/–/p53^+/– (Xpa/p53) mice were exposed up to 14 days to compounds of three distinct classes: genotoxic carcinogens (GTXC), non-genotoxic carcinogens (NGTXC) and non-carcinogens. Subsequently, extensive transcriptomics analyses were performed on several tissues, and transcriptomics data were screened for potential biomarkers using advanced statistical learning techniques. For all tissues analyzed, we identified multigene gene-expression signatures that are, with a high confidence, predictive for GTXC and NGTXC exposures in both mouse genotypes. Xpa/p53 mice did not perform better in the short-term bioassay. We were able to achieve a proof-of-principle for the identification and use of transcriptomics biomarkers for GTXC or NGTXC. This supports the view that toxicogenomics with short-term in vivo exposure provides a viable tool for classifying (geno)toxic compounds.

Induction of neoplastic transformation by ectopic expression of human aldo-keto reductase 1C isoforms in NIH3T3 cells

Chien, C.-W., Ho, I-C., Lee, T.-C. — Mon, 05 Oct 2009 17:18:03 PDT

We have shown previously that chronic low-dose arsenic exposure induces malignant transformation of human skin keratinocyte HaCaT cells. In this study, we found that several isoforms of aldo-keto reductase 1C (AKR1C) were overexpressed in arsenic-exposed HaCaT cells. The AKR1C family of proteins are phase I drug-metabolizing enzymes involved in maintenance of steroid homeostasis, prostaglandin metabolism and metabolic activation of polycyclic aromatic hydrocarbons. To explore the oncogenic potential of AKR1C isoforms, we established mouse NIH3T3 cell lines ectopically and stably expressing human AKR1C1, AKR1C2 or AKR1C3. Our results showed that ectopic expression of human AKR1C1 and AKR1C2, but not AKR1C3, significantly enhanced foci formation. Following subcutaneous injection of these stable cell lines into nude mice, fibrosarcoma were formed from all three cell lines. However, the number and size of tumors formed by the AKR1C3-expressing cell line was fewer and smaller, respectively, than those formed by AKR1C1- and AKR1C2-expressing cells. Inhibitors of AKR1C, genistein and ursodeoxycholic acid, decreased foci formation in AKR1C1- and AKR1C2-expressing NIH3T3 cells in a dose-dependent manner, implying the association of enzymatic activity and oncogenic potential of AKR1C. The requirement of enzymatic ability for neoplastic transformation was confirmed by establishing a NIH3T3 cell line stably expressing a mutant AKR1C1 lacking enzymatic activity, which did not form foci in culture or tumors in nude mice. Our present study reveals that AKR1C enzymatic activity plays crucial roles on induction of neoplastic transformation of mouse NIH3T3 cells.

Backmatter

Tue, 01 Sep 2009 15:11:18 PDT

Frontmatter

Tue, 01 Sep 2009 15:11:18 PDT

Enhanced genomic instabilities caused by deregulated microtubule dynamics and chromosome segregation: a perspective from genetic studies in mice

Rao, C. V., Yamada, H. Y., Yao, Y., Dai, W. — Tue, 01 Sep 2009 15:11:18 PDT

Aneuploidy is defined as numerical abnormalities of chromosomes and is frequently (>90%) present in solid tumors. In general, tumor cells become increasingly aneuploid with tumor progression. It has been proposed that enhanced genomic instability at least contributes significantly to, if not requires, tumor progression. Two major modes for genomic instability are microsatellite instability (MIN) and chromosome instability (CIN). MIN is associated with DNA-level defects (e.g. mismatch repair defects), and CIN is associated with mitotic errors such as chromosome mis-segregation. The mitotic spindle assembly checkpoint (SAC) ensures that cells with defective mitotic spindles or defective interaction between the spindles and kinetochores do not initiate chromosomal segregation during mitosis. Thus, the SAC functions to protect the cell from chromosome mis-segregation and anueploidy during cell division. A loss of the SAC function results in gross aneuploidy, a condition from which cells with an advantage for proliferation will be selected. During the past several years, a flurry of genetic studies in mice and humans strongly support the notion that an impaired SAC causes enhanced genomic instabilities and tumor development. This review article summarizes the roles of key spindle checkpoint proteins {i.e. Mad1/Mad1L1, Mad2/Mad2L1, BubR1/Bub1B, Bub3/Bub3 [conventional protein name (yeast or human)/mouse protein name]} and the modulators (i.e. Chfr/Chfr, Rae1/Rae1, Nup98/Nup98, Cenp-E/CenpE, Apc/Apc) in genomic stability and suppression of tumor development, with a focus on information from genetically engineered mouse model systems. Further elucidation of molecular mechanisms of the SAC signaling has the potential for identifying new targets for rational anticancer drug design.

Over-expression of EphB3 enhances cell-cell contacts and suppresses tumor growth in HT-29 human colon cancer cells

Chiu, S.-T., Chang, K.-J., Ting, C.-H., Shen, H.-C., Li, H., Hsieh, F.-J. — Tue, 01 Sep 2009 15:11:18 PDT

Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in Apc^Min/+ mice and EphB-mediated compartmentalization was demonstrated to be a mechanism suppressing colorectal cancer progression; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4–ephrin-A and EphB4–ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3–ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes’ stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression re-organized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical actin cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased transwell migration, increased apoptosis and Ca²⁺-dependent cell–cell adhesion), decreased mesenchymal markers (fibronectin and nuclear β-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL–Rac1, a known epithelial-to-mesenchymal transition signaling pathway. Additionally, cross talk from Wnt signaling potentiated the restoration of epithelial cell polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3–ephrin-B interaction promotes MET by re-establishing epithelial cell–cell junctions and such an MET-promoting effect contributes to EphB3-mediated tumor suppression.

Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor-2-mediated hepatocellular carcinoma cell invasion

Tue, 01 Sep 2009 15:11:18 PDT

The expression of proteinase-activated receptor (PAR)₂ in human hepatocellular carcinoma (HCC) was established by reverse transcription–polymerase chain reaction, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures and HCC tumor tissue. Stimulation of HCC cells with trypsin and the PAR₂-selective activating peptide, 2-furoyl-LIGRLO-NH₂, increased cell invasion across Matrigel. Both effects were blocked by a PAR₂-selective pepducin antagonist peptide (pal-PAR₂) and by PAR₂ silencing with specific small interfering RNA (siRNA). PAR₂-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by downregulation of Met with specific siRNA. The involvement of Met in PAR₂-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR₂-selective agonist peptide, 2-furoyl-LIGRLO-NH₂, stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 mitogen-activated protein Kinases was found to be critical for the PAR₂–Met receptor tyrosine kinase-invasive signaling axis in HCC cells. Our study establishes an important link between the PAR₂ and Met receptor tyrosine kinase signaling in promoting HCC cell invasion.

Dual role of Ski in pancreatic cancer cells: tumor-promoting versus metastasis-suppressive function

Tue, 01 Sep 2009 15:11:18 PDT

Ski used to be defined as an oncogene that contributes to the resistance of tumor cells to transforming growth factor-β (TGF-β)-induced growth arrest. As TGF-β has a dual effect on tumor growth with both tumor-suppressing and -promoting activity depending on the stage of carcinogenesis and the cell type, the precise role of Ski in carcinogenesis remains unclear. In this study, we show that downregulation of Ski through lentivirus-mediated RNA interference decreases tumor growth both in vitro and in vivo, yet promotes cell invasiveness in vitro, and lung metastasis in vivo in the pancreatic cancer cell line SW1990, which contain wild-type Smad4 expression, and the BxPC3 cell line, which is Smad4 deficient. We also show that the downregulation of Ski increases TGF-β-induced transcriptional activity, which is associated with increased TGF-β-dependent Smad2/3 phosphorylation, and results in an altered expression profile of TGF-β-inducible genes involved in metastasis, angiogenesis and cell proliferation and epithelial–mesenchymal transition. Immunohistochemical analysis of specimens from 71 patients with pancreatic adenocarcinoma showed a significant association between overexpression of Ski and decreased patient survival time (P = 0.0024). Our results suggest that Ski may act as a tumor proliferation-promoting factor or as a metastatic suppressor in human pancreatic cancer.

Identification of XAF1 as a novel cell cycle regulator through modulating G2/M checkpoint and interaction with checkpoint kinase 1 in gastrointestinal cancer

Tue, 01 Sep 2009 15:11:18 PDT

Background and aims: X-linked inhibitor of apoptosis-associated factor 1 (XAF1) was first recognized as an antagonist of X-linked inhibitor of apoptosis in suppressing caspase 3 activity. It has lower expression in cancer cells than normal tissue. Overexpression of XAF1 can inhibit cancer cell growth and sensitize tumor necrosis factor-related apoptosis-inducing ligand- or etoposide-induced apoptosis. The aim of this study is to elucidate the mechanism of XAF1 in regulating cell growth. Methods: Stable transfectants of gastrointestinal (GI) cancer cell lines AGS and SW1116 expressing XAF1 and vector control were generated. Cell growth, apoptosis, mitotic status and cell cycle distribution were assessed. The interaction between XAF1 and G₂/M checkpoint proteins was evaluated by immunoblotting, kinase assay and co-immunoprecipitation assay. Mitotic catastrophe was identified by occurrence of aberrant nuclei and centrosomal amplification. Results: Our results showed that overexpression of XAF1 suppressed serum-dependent cancer cell growth, induced mitotic catastrophe and G₂/M cell cycle arrest. Interestingly, XAF1 was predominantly expressed in G₂/M phase after cell cycle synchronization. XAF1 interacted with and activated checkpoint kinase 1 (Chk1), inactivated Cdc25C and lead to inactivation of Cdc2–cyclin B complex. Suppression of Chk1 abrogated XAF1-induced G₂/M arrest. Conclusions: Our findings implicate XAF1 as a novel cell cycle modulator that is recruited in G₂/M phase and thus unravel a novel function pathway of XAF1, suggesting the potential role of XAF1 as the target for the management of GI cancers.

Cisplatin overcomes Bcl-2-mediated resistance to apoptosis via preferential engagement of Bak: critical role of Noxa-mediated lipid peroxidation

Kutuk, O., Arisan, E. D., Tezil, T., Shoshan, M. C., Basaga, H. — Tue, 01 Sep 2009 15:11:18 PDT

Increased expression of antiapoptotic Bcl-2 proteins confers therapeutic resistance in various cancer types. Targeting Bcl-2 proteins by small molecules or activating alternative pathways to bypass Bcl-2-mediated protection to promote apoptosis are two approaches to overcoming therapeutic resistance. Here, we show that cisplatin triggers a Bak-dependent pathway to induce apoptosis in Bcl-2-overexpressing MCF-7 cells. p53-mediated induction of Noxa expression, generation of lipid peroxidation end products and induction of Noxa–Mcl-1 interaction are necessary for this pathway to function. Although Puma is also induced by cisplatin treatment, it is not required for apoptosis. Similarly, reactive oxygen species production by cisplatin did not have any effect on cisplatin-induced apoptosis in MCF-7 Bcl-2 cells. Furthermore, p53 promotes cisplatin-induced apoptosis by directly binding and counteracting Bcl-x_L antiapoptotic function. In conclusion, our findings suggest a novel mode of action for cisplatin to overcome Bcl-2-mediated protection against apoptosis, which requires preferential activation of Bak and p53-mediated upregulation of Noxa protein levels and lipid peroxidation.

Candidate gene approach evaluates association between innate immunity genes and breast cancer risk in Korean women

Tue, 01 Sep 2009 15:11:18 PDT

Objectives: This study was conducted to investigate the role of common variation in innate immunity-related genes as susceptibility factors to breast cancer risk in Korean women. Methods: Total 1536 single-nucleotide polymorphisms (SNPs) in 203 genes were analyzed by Illumina GoldenGate assay in 209 cases and the same numbers of controls. Both SNP and gene-based tests were used to evaluate the association with breast cancer risk. The robustness of results was further evaluated with permutation method, false discovery rate and haplotype analyses. Results: Both SNP and gene-based analyses showed promising associations with breast cancer risk for 17 genes: OR10J3, FCER1A, NCF4, CNTNAP1, CTNNB1, KLKB1, ITGB2, ALOX12B, KLK2, IRAK3, KLK4, STAT6, NCF2, CCL1, C1QR1, MBP and NOS1. The most significant association with breast cancer risk was observed for the OR10J3 SNP (rs2494251, P-value = 1.2 x 10^–4) and FCER1A SNP (rs7548864, P-value = 7.7 x 10^–4). Gene-based permutation and false discovery rate P-values for OR10J3 SNP (rs2494251) with breast cancer risk were also significant (P = 4 x 10^–5 and 0.008, respectively). Haplotype analyses supported these findings that OR10J3 and FCER1A were most significantly associated with risk for breast cancer (P = 2 x 10^–4 and 0.004, respectively). Conclusion: This study suggests that common genetic variants in the OR10J3 and FCER1A be strongly associated with breast cancer risk among Korean women.

Urinary estrogen metabolites in women at high risk for breast cancer

Im, A., Vogel, V. G., Ahrendt, G., Lloyd, S., Ragin, C., Garte, S., Taioli, E. — Tue, 01 Sep 2009 15:11:18 PDT

Objective: This study explored whether average urinary estrogen metabolites in breast cancer high-risk women can be used to identify a subgroup of women at particularly high risk to develop breast cancer, to which prevention strategies should be addressed. Methods: The population consisted of 77 high-risk women, 30 breast cancer patients and 41 controls. All subjects answered a standardized questionnaire; height and weight and spot urine samples were also obtained. Urine hydroxyestrogen metabolites were measured in triplicate by enzyme immunoassay, and the estrogen metabolite ratios for each individual were calculated. Results: The 2:16 OHE ratio (2-hydroxyestrone/16-alpha-hydroxyestrone) in women at high risk for breast cancer was similar to that observed in the breast cancer group (1.76 ± 2.33 versus 1.29 ± 0.80) and lower than in controls (2.47 ± 1.14; P = 0.00). At the multivariate linear regression model, the 2:16 OHE ratio was significantly associated with diagnosis (P = 0.000 for both the high risk and breast cancer group versus the controls) and body mass index (P = 0.005), but not with age (P = 0.604), or smoking history (P = 0.478). Conclusions: This study suggests that lower urinary 2:16 OHE ratios are predictors of breast cancer risk. Profiling estrogen metabolites may identify women who are more probably to develop breast cancer within a population of women with known risk factors and may help to further elucidate the clinical relevance of urinary 2:16 OHE ratios as clinical markers and prognostic indicators in this population.

Effect of folic acid supplementation on the progression of colorectal aberrant crypt foci

Lindzon, G. M., Medline, A., Sohn, K.-J., Depeint, F., Croxford, R., Kim, Y.-I. — Tue, 01 Sep 2009 15:11:18 PDT

Whether or not folic acid supplementation promotes the progression of colorectal preneoplastic lesions to cancer is an important public health issue, given mandatory fortification and widespread supplemental use of folic acid in North America. We investigated the effect of folic acid supplementation on the progression of aberrant crypt foci (ACF), the earliest precursor of colorectal cancer. Male Sprague-Dawley rats (n = 152) were placed on a control diet (2 mg folic acid/kg diet) at weaning and ACF were induced by azoxymethane. Six weeks post-ACF induction, rats were randomized to receive 0, 2, 5 or 8 mg folic acid/kg diet. At 34 weeks of age, rats were killed, and colorectal tumor parameters, plasma folate and homocysteine (a sensitive inverse indicator of tissue folate status) concentrations and rectal epithelial proliferation were determined. Although the number of ACF increased as dietary folic acid levels increased (P = 0.015), the incidence of colorectal tumors did not differ significantly among the four dietary groups. However, tumor multiplicity was positively correlated with dietary folic acid levels (r = 0.32; P = 0.002) and inversely with plasma homocysteine concentrations (r = –0.32; P = 0.005). Tumor burden was positively correlated with dietary folic acid levels (r = 0.35; P = 0.001) and plasma folate concentrations (r = 0.33; P = 0.008) and inversely with plasma homocysteine concentrations (r = –0.42; P < 0.001). Rectal epithelial proliferation was positively correlated with dietary folic acid levels (r = 0.39; P < 0.001) and plasma folate concentrations (r = 0.34; P < 0.001) and inversely with plasma homocysteine concentrations (r = –0.37; P < 0.001). Our data suggest that folic acid supplementation may promote the progression of ACF to colorectal tumors.

(-)-Epigallocatechin gallate downregulates EGF receptor via phosphorylation at Ser1046/1047 by p38 MAPK in colon cancer cells

Tue, 01 Sep 2009 15:11:18 PDT

We previously reported that (–)-epigallocatechin gallate (EGCG) in green tea alters plasma membrane organization and causes internalization of epidermal growth factor receptor (EGFR), resulting in the suppression of colon cancer cell growth. In the present study, we investigated the detailed mechanism underlying EGCG-induced downregulation of EGFR in SW480 colon cancer cells. Prolonged exposure to EGCG caused EGFR degradation. However, EGCG required neither an ubiquitin ligase (c-Cbl) binding to EGFR nor a phosphorylation of EGFR at tyrosine residues, both of which are reportedly necessary for EGFR degradation induced by epidermal growth factor. In addition, EGCG induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), a stress-inducible kinase believed to negatively regulate tumorigenesis, and the inhibition of p38 MAPK by SB203580, a specific p38 MAPK inhibitor, or the gene silencing using p38 MAPK-small interfering RNA (siRNA) suppressed the internalization and subsequent degradation of EGFR induced by EGCG. EGFR underwent a gel mobility shift upon treatment with EGCG and this was canceled by SB203580, indicating that EGCG causes EGFR phosphorylation via p38 MAPK. Moreover, EGCG caused phosphorylation of EGFR at Ser1046/1047, a site that is critical for its downregulation and this was also suppressed by SB203580 or siRNA of p38 MAPK. Taken together, our results strongly suggest that phosphorylation of EGFR at serine 1046/1047 via activation of p38 MAPK plays a pivotal role in EGCG-induced downregulation of EGFR in colon cancer cells.

Negation of the cancer-preventive actions of selenium by over-expression of protein kinase C{varepsilon} and selenoprotein thioredoxin reductase

Gundimeda, U., Schiffman, J. E., Gottlieb, S. N., Roth, B. I., Gopalakrishna, R. — Tue, 01 Sep 2009 15:11:19 PDT

Selenium prevents cancer in some cases but fails to do so in others. Selenium's failure in this respect may be due to the development of resistance to its chemopreventive actions. Selenocompounds induce a variety of cancer-preventive actions in tumor cells, but these actions may be limited by the low concentrations of free selenocompounds able to reach cells from the plasma. Therefore, we have sought to identify the chemopreventive action requiring the lowest concentration of the redox-active form of selenium, methylseleninic acid (MSA). At submicromolar concentrations, MSA inhibited the malignant transformation of RWPE-1 prostate epithelial cells. In contrast, in already transformed prostate cancer cells, selenium in the micromolar range was required to inhibit cell growth and invasion and to induce apoptosis. The role of protein kinase C (PKC) in these cellular processes, especially the moderately selenium-sensitive PKC, was demonstrated using PKC-specific inhibitors and small interfering RNA. PKC levels inversely correlated with cellular sensitivity to MSA. An over-expression of PKC minimized MSA-induced inhibition of RWPE-1 cell transformation and induction of apoptosis. Thioredoxin reductase (TR), a selenoprotein, reversed the MSA-induced inactivation of PKC isoenzymes. High TR expression in advanced prostate cancer cells correlated with resistance to MSA. Furthermore, inhibition of TR by its specific inhibitor, auranofin, resulted in increased sensitivity of prostate cancer cells to MSA. Collectively, these results suggest that the cancer-preventive actions of selenium may be negated both by an over-expression of PKC, which is a redox-sensitive target for MSA, and by the selenoprotein TR, which reverses PKC sulfhydryl redox modification.

Genetic susceptibility to the development and progression of breast cancer associated with polymorphism of cell cycle and ubiquitin ligase genes

Tue, 01 Sep 2009 15:11:19 PDT

Tumor levels of the cell cycle regulators cyclin E and p27 correlate strongly with survival in breast cancer patients and are specifically regulated by the ubiquitin ligases hCDC4 and SKP2. This study was to explore whether genetic susceptibility to breast cancer is associated with polymorphism of these genes and whether gene–gene and gene–risk factor [i.e. full-term pregnancy (FTP)] interactions are important in determining cancer risk. A two-stage case–control study based on single-nucleotide polymorphisms was performed. The first study (560 cases and 1122 controls) was to define the contribution of cell cycle and ubiquitin ligase genes to cancer susceptibility. The second study (926 cases and 923 controls) was to confirm the association identified in the first stage and to map the variant alleles. Increased breast cancer risk was associated with both polymorphism of hCDC4 and a joint effect of cyclin E and hCDC4. These associations were more significant in nulliparous women, and cancer risk associated with a lower number of FTPs was only seen in women with a higher number of high-risk genotypes, providing support for an effect of gene–risk factor interaction in determining susceptibility. Sequence variants of intron 2 in hCDC4 were found to be the most significant polymorphism and high-stage estrogen receptor (ER)-negative patients carrying the homozygous variant genotype manifested significantly poorer survival. This study concludes that polymorphism of hCDC4 is a risk factor for breast cancer development by interacting with either cyclin E or FTP and may also prove useful in predicting progression of patients with high-stage and ER-negative breast cancers.

Characterization of the cancer chemopreventive NRF2-dependent gene battery in human keratinocytes: demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2 pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds

Tue, 01 Sep 2009 15:11:19 PDT

To better understand the role of transcription factor NF-E2-related factor (NRF) 2 in the human and its contribution to cancer chemoprevention, we have knocked down its negative regulators, Kelch-like ECH-associated protein 1 (KEAP1) and broad-complex, tramtrack and bric à brac and cap'n'collar homology 1 (BACH1), in HaCaT keratinocytes. Whole-genome microarray revealed that knockdown of KEAP1 resulted in 23 messenger RNAs (mRNAs) being up-regulated ≥2.0-fold. mRNA for aldo-keto reductase (AKR) 1B10, AKR1C1, AKR1C2 and AKR1C3 were induced to the greatest extent, showing increases of between 12- and 16-fold, whereas mRNA for glutamate-cysteine ligase catalytic and modifier subunits, NAD(P)H:quinone oxidoreductase-1 and haem oxygenase-1 (HMOX1) were induced between 2.0- and 4.8-fold. Knockdown of BACH1 increased HMOX1 135-fold but induced the other genes examined to a maximum of only 2.7-fold. Activation of NRF2, by KEAP1 knockdown, caused a 75% increase in the amount of glutathione in HaCaT cells and a 1.4- to 1.6-fold increase in their resistance to the electrophiles acrolein, chlorambucil and cumene hydroperoxide (CuOOH), as well as the redox-cycling agent menadione. Inhibition of glutathione synthesis during KEAP1 knockdown, by treatment with buthionine sulfoximine, abrogated resistance to acrolein, chlorambucil and CuOOH, but not to menadione. In contrast, knockdown of BACH1 did not increase glutathione levels or resistance to xenobiotics. Knockdown of NRF2 in HaCaT cells decreased glutathione to ~80% of normal homeostatic levels and similarly reduced their tolerance of electrophiles. Thus, the KEAP1–NRF2 pathway determines resistance to electrophiles and redox-cycling compounds in human keratinocytes through glutathione-dependent and glutathione-independent mechanisms. This study also shows that AKR1B10, AKR1C1 and AKR1C2 proteins have potential utility as biomarkers for NRF2 activation in the human.

Disruption of estrogen receptor signaling enhances intestinal neoplasia in ApcMin/+ mice

Tue, 01 Sep 2009 15:11:19 PDT

Estrogen receptors (ERs) [ER (Esr1) and ERβ (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ER and ERβ is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ER knockout and Apc^Min mouse strains, we demonstrate that ER deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc^Min/+ mice. Within the normal intestinal epithelium of Apc^Min/+ mice, ER deficiency is associated with an accumulation of nuclear β-catenin, an indicator of activation of the Wnt–β-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ER deficiency is associated with activation of Wnt–β-catenin signaling, ER deficiency in the intestinal epithelium of Apc^Min/+ mice also correlated with increased expression of Wnt–β-catenin target genes. Through crosses between an ERβ knockout and Apc^Min mouse strains, we observed some evidence that ERβ deficiency is associated with an increased incidence of colon tumors in Apc^Min/+ mice. This effect of ERβ deficiency does not involve modulation of Wnt–β-catenin signaling. Our studies suggest that ER and ERβ signaling modulate colorectal carcinogenesis, and ER does so, at least in part, by regulating the activity of the Wnt–β-catenin pathway.

Genetic mapping of Mom5, a novel modifier of ApcMin-induced intestinal tumorigenesis

Tue, 01 Sep 2009 15:11:19 PDT

The initial purpose of this study was to assess the role of estrogen receptor β (ERβ) in intestinal tumorigenesis by examining the effects of an ERβ knockout (ERβ^–/–) on Apc^Min mice. In order to accomplish this goal on a uniform genetic background, we were required to backcross the ERβ knockout from the 129P2 genetic background to the B6 genetic background for 10 generations. Midway through this process, we performed a test cross in which mice from the N₅ backcross generation of the ERβ knockout strain were intercrossed with Apc^Min/+ mice to obtain Apc^Min/+ ERβ^+/+, Apc^Min/+ ERβ^+/– and Apc^Min/+ ERβ^–/– mice. Intestinal tumorigenesis in the N₅F₂ mice was evaluated at 14 weeks of age. The analysis of the impact of ERβ in the N₅ cross was complicated by segregating 129P2-derived alleles that affected tumor number and were unlinked to ERβ. Genetic linkage analysis of this cross permitted the localization of a single genetic modifier of tumor number in Apc^Min/+ mice. This locus, Modifier of Min 5 (Mom5), maps to proximal mouse chromosome 5; the 129P2 allele of this locus is associated with a 50% reduction in mean intestinal tumor number. Through in silico analysis and confirmatory sequencing, we have identified the Rad50-interacting protein-1 gene as a strong candidate for Mom5.

Deficient deletion of apoptotic cells by macrophage migration inhibitory factor (MIF) overexpression accelerates photocarcinogenesis

Tue, 01 Sep 2009 15:11:19 PDT

Chronic ultraviolet (UV) exposure can increase the occurrence of p53 mutations, thus leading to a dysregulation of apoptosis and the initiation of skin cancer. Therefore, it is extremely important that apoptosis is induced quickly after UV irradiation, without any dysregulation. Recent studies have suggested a potentially broader role for macrophage migration inhibitory factor (MIF) in growth regulation via its ability to antagonize p53-mediated gene activation and apoptosis. To further elucidate the possible role of MIF in photocarcinogenesis, the acute and chronic UVB effect in the skin was examined using macrophage migration inhibitory factor transgenic (MIF Tg) and wild-type (WT) mice. The MIF Tg mice exposed to chronic UVB irradiation began to develop skin tumors after ~14 weeks, whereas the WT mice began to develop tumors after 18 weeks. A higher incidence of tumors was observed in the MIF Tg in comparison with the WT mice after chronic UVB irradiation. Next, we clarified whether the acceleration of photo-induced carcinogenesis in the MIF Tg mice was mediated by the inhibition of apoptosis There were fewer sunburned cells in the epidermis of the MIF Tg mice than the WT mice after acute UVB exposure. The epidermis derived from the MIF Tg mice exhibited substantially decreased levels of p53, bax and p21 after UVB exposure in comparison with the WT mice. Collectively, these findings suggest that chronic UVB exposure enhances MIF production, which may inhibit the p53-dependent apoptotic processes and thereby induce photocarcinogenesis in the skin.

Activation of thromboxane A2 receptors induces orphan nuclear receptor Nurr1 expression and stimulates cell proliferation in human lung cancer cells

Li, X., Tai, H.-H. — Tue, 01 Sep 2009 15:11:19 PDT

Previous studies implicate that activation of thromboxane A₂ receptor (TP) induced cell proliferation and transformation in several cell lines. We report here that the activation of TP by its agonist, [1S-[1, 2 (Z), 3β (1E, 3S*), 4]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo [2.2.1] hept-2-yl]-5-heptenoic acid (I-BOP), induced Nurr1 expression and stimulated proliferation of human lung cancer cells. Nurr1, an orphan nuclear receptor in the nuclear receptor subfamily 4A subfamily, has been implicated in cell proliferation, differentiation and apoptosis. I-BOP markedly induced Nurr1 messenger RNA and protein levels as compared with other subfamily members, Nur77 and Nor-1. The signaling pathways of I-BOP-induced Nurr1 expression were examined by using various inhibitors of signaling molecules. The induction of Nurr1 expression by I-BOP appeared to be mediated through protein kinase A (PKA)/cAMP response element binding (CREB), protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways and not related to epidermal growth factor receptor and prostaglandin E₂ pathways. Transcriptional activation of Nurr1 gene by I-BOP was further investigated at the promoter level in H157 cells. 5'-Deletion analysis, site-directed mutagenesis and luciferase reporter assay demonstrated that Nurr1 expression was induced by I-BOP in a PKA/CREB-dependent manner. Further studies have revealed that Nurr1 may mediate cyclin D1 expression and I-BOP-induced cell proliferation in H157 cells since small interfering RNA of Nurr1 blocked I-BOP-induced cyclin D1 expression and cell proliferation and also decreased cell growth rate. These results provide strong evidence that Nurr1 plays a significant role in cell proliferation and may mediate TP agonist-induced proliferation in lung cancer cells.

Role of NKX2-1 in N-bis(2-hydroxypropyl)-nitrosamine-induced thyroid adenoma in mice

Tue, 01 Sep 2009 15:11:19 PDT

NKX2-1 is a homeodomain transcription factor that is critical for genesis of the thyroid and transcription of the thyroid-specific genes. Nkx2-1-thyroid-conditional hypomorphic mice were previously developed in which Nkx2-1 gene expression is lost in 50% of the thyroid cells. Using this mouse line as compared with wild-type and Nkx2-1 heterozygous mice, a thyroid carcinogenesis study was carried out using the genotoxic carcinogen N-bis(2-hydroxypropyl)-nitrosamine (DHPN), followed by sulfadimethoxine (SDM) or the non-genotoxic carcinogen amitrole (3-amino-1,2,4-triazole). A significantly higher incidence of adenomas was obtained in Nkx2-1-thyroid-conditional hypomorphic mice as compared with the other two groups of mice only when they were treated with DHPN + SDM, but not amitrole. A bromodeoxyuridine incorporation study revealed that thyroids of the Nkx2-1-thyroid-conditional hypomorphic mice had >2-fold higher constitutive cell proliferation rate than the other two groups of mice, suggesting that this may be at least partially responsible for the increased incidence of adenoma in this mouse line after genotoxic carcinogen exposure. Thus, NKX2-1 may function to control the proliferation of thyroid follicular cells following damage by a genotoxic carcinogen.

The prostaglandin receptor EP2 activates multiple signaling pathways and {beta}-arrestin1 complex formation during mouse skin papilloma development

Chun, K.-S., Lao, H.-C., Trempus, C. S., Okada, M., Langenbach, R. — Tue, 01 Sep 2009 15:11:19 PDT

Prostaglandin E₂ (PGE₂) is elevated in many tumor types, but PGE₂'s contributions to tumor growth are largely unknown. To investigate PGE₂'s roles, the contributions of one of its receptors, EP2, were studied using the mouse skin initiation/promotion model. Initial studies indicated that protein kinase A (PKA), epidermal growth factor receptor (EGFR) and several effectors—cyclic adenosine 3',5'-monophosphate response element-binding protein (CREB), H-Ras, Src, protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2—were activated in 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted papillomas and that PKA and EGFR inhibition (H89 and AG1478, respectively) decreased papilloma formation. EP2's contributions to the activation of these pathways and papilloma development were determined by inhibiting endogenous TPA-induced PGE₂ production with indomethacin (Indo) and concomitantly treating with the EP2 agonist, CAY10399 (CAY). CAY treatment restored papilloma formation in TPA/Indo-treated mice and increased cyclic adenosine 3',5'-monophosphate and PKA activation as measured by p-CREB formation. CAY treatment also increased EGFR and Src activation and their inhibition by AG1478 and PP2 indicated that Src was upstream of EGFR. CAY also increased H-Ras, ERK1/2 and AKT activation, and AG1478 decreased their activation indicating EGFR being upstream. Supporting EP2's contribution, EP2–/– mice exhibited 65% fewer papillomas and reduced Src, EGFR, H-Ras, AKT and ERK1/2 activation. G protein-coupled receptor (GPCR) activation of EGFR has been reported to involve Src's activation via a GPCR–β-arrestin–Src complex. Indeed, immunoprecipitation of β-arrestin1 or p-Src indicated the presence of an EP2–β-arrestin1–p-Src complex in papillomas. The data indicated that EP2 contributed to tumor formation via activation of PKA and EGFR and that EP2 formed a complex with β-arrestin1 and Src that contributed to signaling and/or EP2 desensitization.

Etoposide induces MLL rearrangements and other chromosomal abnormalities in human embryonic stem cells

Tue, 01 Sep 2009 15:11:19 PDT

MLL rearrangements are hallmark genetic abnormalities in infant leukemia known to arise in utero. They can be induced during human prenatal development upon exposure to etoposide. We also hypothesize that chronic exposure to etoposide might render cells more susceptible to other genomic insults. Here, for the first time, human embryonic stem cells (hESCs) were used as a model to test the effects of etoposide on human early embryonic development. We addressed whether: (i) low doses of etoposide promote MLL rearrangements in hESCs and hESCs-derived hematopoietic cells; (ii) MLL rearrangements are sufficient to confer hESCs with a selective growth advantage and (iii) continuous exposure to low doses of etoposide induces hESCs to acquire other chromosomal abnormalities. In contrast to cord blood-derived CD34⁺ and hESC-derived hematopoietic cells, exposure of undifferentiated hESCs to a single low dose of etoposide induced a pronounced cell death. Etoposide induced MLL rearrangements in hESCs and their hematopoietic derivatives. After long-term culture, the proportion of hESCs harboring MLL rearrangements diminished and neither cell cycle variations nor genomic abnormalities were observed in the etoposide-treated hESCs, suggesting that MLL rearrangements are insufficient to confer hESCs with a selective proliferation/survival advantage. However, continuous exposure to etoposide induced MLL breaks and primed hESCs to acquire other major karyotypic abnormalities. These data show that chronic exposure of developmentally early stem cells to etoposide induces MLL rearrangements and make hESCs more prone to acquire other chromosomal abnormalities than postnatal CD34⁺ cells, linking embryonic genotoxic exposure to genomic instability.

Multidirectional tumor-suppressive activity of AIMP2/p38 and the enhanced susceptibility of AIMP2 heterozygous mice to carcinogenesis

Choi, J. W., Um, J. Y., Kundu, J. K., Surh, Y.-J., Kim, S. — Tue, 01 Sep 2009 15:11:19 PDT

Aminoacyl-transfer ribonucleic acid (tRNA) synthetases-interacting multifunctional protein (AIMP) 2 is a factor associated with the macromolecular protein synthesis machinery consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors. However, it was shown to work as a multifaceted regulator through the versatile interactions with diverse signal mediators. For instance, it can mediate pro-apoptotic response to DNA damage and tumor necrosis factor- (TNF-) stimulus and growth-arresting signal by transforming growth factor (TGF)-β. Considering that these pathways are critically implicated in the control of tumorigenesis, AIMP2 is expected to work as a potent tumor suppressor with broad coverage against different cancer types. Here we investigated whether AIMP2 would give gene dosage effect on its pro-apoptotic and anti-proliferative activities using the wild-type, hetero- and homozygous AIMP2 cells and whether AIMP2 would be critical in preventing tumorigenesis using different in vivo tumor models. Both the apoptotic responses to DNA damage and TNF- and sensitivity to growth arresting TGF-β signal were reduced in AIMP2 hetero- and homozygous cells compared with the wild-type cells in dose-dependent manner. In all the in vivo carcinogenesis experiments, reduction of AIMP2 level in heterozygous AIMP2 mice provided higher susceptibility to tumor formation. Thus, this work proves the functional significance of AIMP2 in determination of cell proliferation and death, and as a haploinsufficient tumor suppressor.

Dual role for Id2 in chemical carcinogen-induced skin tumorigenesis

Tue, 01 Sep 2009 15:11:19 PDT

Inhibitor of DNA binding 2 (Id2) is a negative regulator of basic helix-loop-helix transcription factors and is involved in the control of cellular differentiation and proliferation. By using a two-step chemical carcinogenesis protocol, we evaluated the role of Id2 in skin tumor formation in mice. Twenty weeks after the initiation, the number of tumors formed in the Id2^–/– mice was 3.5-fold higher than that in their wild-type littermates, whereas the diameter of tumors in the Id2^–/– mice was about half of that of the tumors in the wild-type mice. In the Id2^–/– mice, epidermal T cells, which play a key role in immunosurveillance against skin tumor development, were barely detectable. Although histological analyses demonstrated no apparent difference in tumor cell type, tumor vessel formation or apoptosis, the proportion of proliferating cells was reduced in the tumors in the Id2^–/– mice compared with those in the wild-type mice. In the wild-type mice, the expression of Id2 was enhanced in skin tumors compared with that in ear epidermal cells. Biochemical analysis demonstrated that cyclin D1 was reduced at the protein level in the tumors in the Id2^–/– mice, whereas other factors such as cyclin E and p27 were not altered significantly. Our results reveal that Id2 plays a dual role in skin tumorigenesis by suppressing tumor development through the establishment of epidermal T cell-mediated skin immunosurveillance and by promoting tumor cell proliferation via the control of the cyclin D1 protein level.

Backmatter

Wed, 29 Jul 2009 18:32:35 PDT

Frontmatter

Wed, 29 Jul 2009 18:32:35 PDT

Metabolic transformation in cancer

Tennant, D. A., Duran, R. V., Boulahbel, H., Gottlieb, E. — Wed, 29 Jul 2009 18:32:35 PDT

In 2000, Douglas Hanahan and Robert Weinberg published a review detailing the six hallmarks of cancer. These are six phenotypes that a tumour requires in order to become a fully fledged malignancy: persistent growth signals, evasion of apoptosis, insensitivity to anti-growth signals, unlimited replicative potential, angiogenesis and invasion and metastasis. However, it is becoming increasingly clear that these phenotypes do not portray the whole story and that other hallmarks are necessary: one of which is a shift in cellular metabolism. The tumour environment creates a unique collection of stresses to which cells must adapt in order to survive. This environment is formed by the uncontrolled proliferation of cells, which ignore the cues that would create normal tissue architecture. As a result, the cells forming the tumour are exposed to low oxygen and nutrient levels, as well as high levels of toxic cellular waste products, which is thought to propel cells towards a more transformed phenotype, resistant to cell death and pro-metastatic.

The type III transforming growth factor-{beta} receptor negatively regulates nuclear factor kappa B signaling through its interaction with {beta}-arrestin2

You, H. J., How, T., Blobe, G. C. — Wed, 29 Jul 2009 18:32:35 PDT

Transforming growth factor-β (TGF-β) increases or decreases nuclear factor kappa B (NFB) signaling in a context-dependent manner through mechanisms that remain to be defined. The type III transforming growth factor-β receptor (TβRIII) is a TGF-β superfamily co-receptor with emerging roles in both mediating and regulating TGF-β superfamily signaling. We have previously reported a novel interaction of TβRIII with the scaffolding protein, β-arrestin2, which results in TβRIII internalization and downregulation of TGF-β signaling. β-arrestin2 also scaffolds interacting receptors with the mitogen-activated protein kinase and NFB-signaling pathways. Here, we demonstrate that TβRIII, through its interaction with β-arrestin2, negatively regulates NFB signaling in MCF10A breast epithelial and MDA-MB-231 breast cancer cells. Increasing TβRIII expression reduced NFB-mediated transcriptional activation and IB degradation, whereas a TβRIII mutant unable to interact with β-arrestin2, TβRIII-T841A, had no effect. In a reciprocal manner, short hairpin RNA-mediated silencing of either TβRIII expression or β-arrestin2 expression increased NFB-mediated transcriptional activation and IB degradation. Functionally, TβRIII-mediated repression of NFB signaling is important for TβRIII-mediated inhibition of breast cancer cell migration. These studies define a mechanism through which TβRIII regulates NFB signaling and expand the roles of this TGF-β superfamily co-receptor in regulating epithelial cell homeostasis.

Proteomic approach to ETV5 during endometrial carcinoma invasion reveals a link to oxidative stress

Wed, 29 Jul 2009 18:32:35 PDT

Endometrial cancer, the most common gynecological malignancy in western countries, is characterized by a favorable prognosis. Nonetheless, deep myometrial invasion correlates with more undifferentiated tumors, lymph-vascular invasion, node involvement and decreased global survival. We have described previously the Ets family member ERM/ETV5 specifically upregulated in endometrial endometrioid carcinoma (EEC) associated with myometrial infiltration. To understand the role of this transcription factor during myometrial infiltration, we analyzed by two-dimension differential gel electrophoresis (2D-DIGE) technology those proteins whose expression was altered in endometrial cell lines stably overexpressing ERM/ETV5. Pathway analysis pointed to actin regulation and transforming growth factor beta and progesterone signaling as processes regulated by ERM/ETV5. In addition, we characterized the specific upregulation of the nuclear dehydrogenase/reductase Hep27 as well as its ERM/ETV5-dependent mitochondrial localization. Further functional studies demonstrated a protective role of Hep 27 against apoptosis induced by oxidative stress. Overall, the ETV5-related proteomic approach performed in the Hec-1A cell line reinforces a role of this transcription factor in the regulation of the migratory and invasive tumor behavior and points to a modulated response to oxidative stress associated with the promotion of invasion in endometrial cancer. Unraveling the molecular events in EEC associated with the initiation of tumor invasion would represent an obvious improvement in the pursuit of rational targets for the onset of metastasis. This knowledge would also be a valuable tool for the molecular stratification of patients since myometrial affectation determines an increase in the rate of recurrence after a first surgical treatment and a decrease in 5 year survival.

Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase

Wed, 29 Jul 2009 18:32:35 PDT

Resistance to chemotherapy is a major obstacle for successful treatment of breast cancer patients. Given that prolactin (PRL) acts as an anti-apoptotic/survival factor in the breast, we postulated that it antagonizes cytotoxicity by chemotherapeutic drugs. Treatment of breast cancer cells with PRL caused variable resistance to taxol, vinblastine, doxorubicin and cisplatin. PRL prevented cisplatin-induced G₂/M cell cycle arrest and apoptosis. In the presence of PRL, significantly less cisplatin was bound to DNA, as determined by mass spectroscopy, and little DNA damage was seen by -H2AX staining. PRL dramatically increased the activity of glutathione-S-transferase (GST), which sequesters cisplatin in the cytoplasm; this increase was abrogated by Jak and mitogen-activated protein kinase inhibitors. PRL upregulated the expression of the GSTµ, but not the , isozyme. A GST inhibitor abrogated antagonism of cisplatin cytotoxicity by PRL. In conclusion, PRL confers resistance against cisplatin by activating a detoxification enzyme, thereby reducing drug entry into the nucleus. These data provide a rational explanation for the ineffectiveness of cisplatin in breast cancer, which is characterized by high expression of both PRL and its receptor. Suppression of PRL production or blockade of its actions should benefit patients undergoing chemotherapy by allowing for lower drug doses and expanded drug options.

Insulin-like growth factor-I receptor blockade reduces the invasiveness of gastrointestinal cancers via blocking production of matrilysin

Wed, 29 Jul 2009 18:32:35 PDT

Insulin-like growth factor-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal (GI) cancers. We have previously shown significant therapeutic activity for recombinant adenoviruses expressing dominant-negative insulin-like growth factor-I receptor (IGF-IR/dn), including suppression of tumor invasion. In this study, we sought to evaluate the mechanism of inhibition of invasion and the relationship between IGF-IR and matrix metalloproteinase (MMP) activity in GI carcinomas. We analyzed the role of IGF-IR on invasion in three GI cancer cell lines, colorectal adenocarcinoma, HT29; pancreatic adenocarcinoma, BxPC3 and gastric adenocarcinoma, MKN45, using a modified Boyden chamber method and subcutaneous xenografts in nude mice. The impact of IGF-IR signaling on the expression of MMPs and the effects of blockade of matrilysin or IGF-IR on invasiveness were assessed using recombinant adenoviruses, a tyrosine kinase inhibitor NVP-AEW541 and antisense matrilysin. Invasive subcutaneous tumors expressed several MMPs. IGF-IR/dn reduced the expression of these MMPs but especially matrilysin (MMP-7). Insulin-like growth factor (IGF) stimulated secretion of matrilysin and IGF-IR/dn blocked IGF-mediated matrilysin induction in three GI cancers. Both IGF-IR/dn and inhibition of matrilysin reduced in vitro invasion to the same degree. NVP-AEW541 also reduced cancer cell invasion both in vitro and in murine xenograft tumors via suppression of matrilysin. Thus, blockade of IGF-IR is involved in the suppression of cancer cell invasion through downregulation of matrilysin. Strategies of targeting IGF-IR may have significant therapeutic utility to prevent invasion and progression of human GI carcinomas.

Human RIF1 encodes an anti-apoptotic factor required for DNA repair

Wed, 29 Jul 2009 18:32:35 PDT

Human Rap1-interacting protein 1 (RIF1) contributes to the ataxia telangiectasia, mutated-mediated DNA damage response against the dexterous effect of DNA lesions and plays a critical role in the S-phase checkpoint. However, the molecular mechanisms by which human RIF1 conquers DNA aberrations remain largely unknown. We here showed that inhibition of RIF1 expression by small interfering RNA led to defective homologous recombination-mediated DNA double-strand break repair and sensitized cancer cells to camptothecin or staurosporine treatment. RIF1 underwent caspase-dependent cleavage upon apoptosis. We further found that RIF1 was highly expressed in human breast tumors, and its expression status was positively correlated with differentiation degrees of invasive ductal carcinoma of the breast. Our results suggest that RIF1 encodes an anti-apoptotic factor required for DNA repair and is a potential target for cancer treatment.

Triggering of transient receptor potential vanilloid type 1 (TRPV1) by capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ATM-dependent manner

Wed, 29 Jul 2009 18:32:35 PDT

Herein, we provide evidence on the expression of transient receptor potential vanilloid type 1 (TRPV1) on human urothelial cancer (UC) cells and its involvement in the apoptosis induced by the selective agonist capsaicin (CPS). We analyzed TRPV1 messenger RNA and protein expression on human UC cell lines demonstrating its progressive decrease in high-grade UC cells. Treatment of RT4 cells with CPS induced cell cycle arrest in G₀/G₁ phase and apoptosis. These events were associated with rapid co-ordinated transcription of pro-apoptotic genes including Fas/CD95, Bcl-2 and caspase families and ataxia telangiectasia mutated (ATM)/CHK2/p53 DNA damage response pathway. CPS induced Fas/CD95 upregulation, but more importantly Fas/CD95 ligand independent, TRPV1-dependent death receptor clustering and triggering of both extrinsic and intrinsic mitochondrial-dependent pathways. Moreover, we observed that CPS activates ATM kinase that is involved in Ser15, Ser20 and Ser392 p53 phosphorylation as shown by the use of the specific inhibitor KU55933. Notably, ATM activation was also found to control upregulation of Fas/CD95 expression and its co-clustering with TRPV1 as well as RT4 cell growth and apoptosis. Altogether, we describe a novel connection between ATM DNA damage response pathway and Fas/CD95-mediated intrinsic and extrinsic apoptotic pathways triggered by TRPV1 stimulation on UC cells.

Differential repetitive DNA methylation in multiple myeloma molecular subgroups

Wed, 29 Jul 2009 18:32:35 PDT

Multiple myeloma (MM) is characterized by a wide spectrum of genetic changes. Global hypomethylation of repetitive genomic sequences such as long interspersed nuclear element 1 (LINE-1), Alu and satellite alpha (SAT-) sequences has been associated with chromosomal instability in cancer. Methylation status of repetitive elements in MM has never been investigated. In the present study, we used a quantitative bisulfite-polymerase chain reaction pyrosequencing method to evaluate the methylation patterns of LINE-1, Alu and SAT- in 23 human myeloma cell lines (HMCLs) and purified bone marrow plasma cells from 53 newly diagnosed MM patients representative of different molecular subtypes, 7 plasma cell leukemias (PCLs) and 11 healthy controls. MMs showed a decrease of Alu [median: 21.1 %5-methylated cytosine (%5mC)], LINE-1 (70.0%5mC) and SAT- (77.9%5mC) methylation levels compared with controls (25.2, 79.5and 89.5%5mC, respectively). Methylation levels were lower in PCLs and HMCLs compared with MMs (16.7 and 14.8%5mC for Alu, 45.5 and 42.4%5mC for LINE-1 and 33.3 and 43.3%5mC for SAT-, respectively). Notably, LINE-1 and SAT- methylation was significantly lower in the non-hyperdiploid versus hyperdiploid MMs (P = 0.01 and 0.02, respectively), whereas Alu and SAT- methylation was significantly lower in MMs with t(4;14) (P = 0.02 and 0.004, respectively). Finally, we correlated methylation patterns with DNA methyltransferases (DNMTs) messenger RNA levels showing in particular a progressive and significant increase of DNMT1 expression from controls to MMs, PCLs and HMCLs (P < 0.001). Our results indicate that global hypomethylation of repetitive elements is significantly associated with tumor progression in MM and may contribute toward a more extensive stratification of the disease.

Interferon-beta treatment increases human papillomavirus early gene transcription and viral plasmid genome replication by activating interferon regulatory factor (IRF)-1

Wed, 29 Jul 2009 18:32:35 PDT

Interferons (IFNs) have been used to treat mucosal lesions caused by human papillomavirus (HPV) infection, such as intraepithelial precursor lesions to cancer of the uterine cervix, genital warts or recurrent respiratory papillomatosis, to potentially reduce or eliminate replicating HPV plasmid genomes. Mucosal HPVs have evolved mechanisms that impede IFN-β synthesis and downregulate genes induced by IFN. Here we show that these HPV types directly subvert a cellular transcriptional response to IFN-β as a potential boost in infection. Treatment with low levels of human IFN-β induced initial amplification of HPV-16 and HPV-11 plasmid genomes and increased HPV-16 or HPV-31 DNA copy numbers up to 6-fold in HPV-immortalized keratinocytes. IFN treatment also increased early gene transcription from the major early gene promoters in HPV-16, HPV-31 and HPV-11. Furthermore, mutagenesis of the viral genomes and ectopic interferon regulatory factor (IRF) expression in transfection experiments using IRF-1^–/–, IRF-2^–/– and dual knockout cell lines determined that these responses are due to the activation of IRF-1 interaction with a conserved interferon response element demonstrated in several mucosal HPV early gene promoters. Our results provide a molecular explanation for the varying clinical outcomes of IFN therapy of papillomatoses and define an assay for the modulation of the HPV gene program by IFNs as well as other cytokines and signaling molecules in infection and therapy.

Altered expression of the human base excision repair gene NTH1 in gastric cancer

Wed, 29 Jul 2009 18:32:35 PDT

A base excision repair enzyme, NTH1, has activity that is capable of removing oxidized pyrimidines, such as thymine glycol (Tg), from DNA. To clarify whether the NTH1 gene is involved in gastric carcinogenesis, we first examined the NTH1 expression level in eight gastric cancer cell lines, and the results showed that NTH1 expression was downregulated in all of them, including cell line AGS. Next, a comparison of excisional repair activity against Tg by empty vector-transfected AGS clones and FLAG-NTH1-expressing AGS clones showed that a low NTH1 expression level led to low capacity to repair the damaged base in the gastric epithelial cells. Reduced messenger RNA expression of NTH1 was also detected in 36% (18/50) of primary gastric cancers. Moreover, immunohistochemical analysis revealed that NTH1 was predominantly localized in the cytoplasm in 24% (12/50) of the primary gastric cancers in contrast to the nuclear localization in non-cancerous tissue, suggesting impaired excisional repair ability for nuclear DNA. No associations between clinicopathological factors and NTH1 expression level or localization pattern were detected in the gastric cancers. Next, we found two novel genetic polymorphisms, i.e. c.-163C>G and c.-241_-221del, in the NTH1 promoter region, and a luciferase assay showed that both were associated with reduced promoter activity. However, there were no associations between the polymorphisms and risk of gastric cancer in a gastric cancer case–control study. These findings suggested that downregulation of NTH1 expression and abnormal localization of NTH1 may be involved in the pathogenesis of a subset of gastric cancers.

Novel single nucleotide polymorphism associations with colorectal cancer on chromosome 8q24 in African and European Americans

Wed, 29 Jul 2009 18:32:35 PDT

Regions on chromosome 8q24 harbor susceptibility alleles for multiple cancers including colorectal (region 3) and prostate cancer (regions 1–4). The objectives of the present study were (i) to test whether single-nucleotide polymorphisms (SNPs) in region 4 are associated with colorectal cancer (CRC) in European or African Americans; (ii) to test whether 8q24 SNPs previously shown to be associated with colorectal and prostate cancer also show association in our multiethnic series and (iii) to test for association between 100 ancestry informative markers (AIMs) and CRC in both the African American and European American cohorts. In total, we genotyped nine markers on 8q24 and 100 unlinked AIMs in 569 CRC cases and 439 controls (490 European Americans and 518 African Americans) obtained retrospectively from a hospital-based sample. We found rs7008482 in 8q24 region 4 to be significantly associated with CRC in European Americans (P = 0.03). Also in region 4, we found that a second SNP, rs16900305, trended toward association with CRC in African Americans. The rs6983267 in region 3, previously implicated in CRC risk, trended toward association with disease in European Americans but not in African Americans. Finally, none of the 100 AIMs tested for association reached statistical significance after correction for multiple hypothesis testing. In summary, these results are evidence that 8q24 region 4 contains novel CRC-associated alleles in European and African Americans.

Cytokine genetic polymorphisms and prostate cancer aggressiveness

Wed, 29 Jul 2009 18:32:35 PDT

Prostate cancer (PCa) is one of the most common cancers in the world. Inflammation has been described as a risk factor for PCa and depends on the production of cytokines in response to tissue damage or the presence of stimuli that induces cellular stress. Interindividual variation in cytokine production is partially controlled by single-nucleotide polymorphisms (SNPs) that have been associated with differential production of cytokines. We have recently showed that SNP–SNP interactions of cytokine genes are associated with PCa risk. However, little is known about the association of cytokine SNPs and PCa aggressiveness. In this study, we evaluated the association of 15 SNPs in five cytokine genes and aggressiveness of PCa in African- and Caucasian-American individuals. Caucasian Americans with the genotypes IL10–1082GG or IL1B+3954TT had 2.31-fold [95% confidence interval (CI) = 1.13–4.72] and 3.11 (95% CI = 1.20–8.06)-fold risk, respectively, of developing aggressive PCa, as compared with individuals without those genotypes. We did not find any associations in the African-American group. Using Multivariate Adaptive Regression Splines modeling for exploratory SNP–SNP interactions, our results showed that more aggressive PCa in Caucasians Americans is associated with the CT genotype at IL8–47 [odds ratios (OR) = 3.50; 95% CI = 1.13–10.88] or combined genotypes of IL1B–511CC and IL10–1082GG (OR = 3.38; 95% CI = 1.70–6.71). Unfortunately, the same analysis could not be performed in the African-Americans due to limited number of individuals. With limited sample size, the results from this study suggest that SNPs in cytokine genes may be associated with PCa aggressiveness. More extensive studies are warranted to validate our findings.

Epidermal growth factor A61G gene polymorphism, gastroesophageal reflux disease and esophageal adenocarcinoma risk

Wed, 29 Jul 2009 18:32:35 PDT

Background: Single-nucleotide polymorphisms of key cancer genes, such as EGF A61G, are associated with an elevated risk of esophageal adenocarcinoma (EAC). As gastroesophageal reflux disease (GERD) is an established risk factor for EAC, we evaluated whether the association between epidermal growth factor (EGF) polymorphism and EAC development is altered by the presence of GERD. Methods: EGF genotyping of DNA samples was performed and GERD history was collected for 309 EAC patients and 275 matched healthy controls. Associations between genotypes and EAC risk were evaluated using adjusted logistic regression. Genotype–GERD relationships were explored using analyses stratified by GERD history and joint effects models that considered severity and duration of GERD symptoms. Results: EGF variants (A/G or G/G) were more common (P = 0.02) and GERD was more prevalent (P < 0.001) in cases than in controls. When compared with the EGF wild-type A/A genotype, the G/G variant was associated with a substantial increase in EAC risk among individuals with GERD [Odds ratio 9.7; 95% confidence interval (CI), 3.8–25.0; P < 0.001] and a slight decrease in risk for GERD-free individuals (odds ratio 0.4; 95% CI = 0.22–0.90; P = 0.02). In the joint effects models, the odds of EAC was also highest for G/G patients (when compared with A/A) who either experienced frequent GERD of more than once per week (odds ratio 21.8; 95% CI = 5.1–94.0; P < 0.001) or suffered GERD for longer than 15 years (odds ratio 22.4; 95% CI = 6.5–77.6; P < 0.001). There was a highly significant interaction between the G/G genotype and the presence of GERD (P < 0.001). Conclusions: EGF A61G polymorphism may alter EAC susceptibility through an interaction with GERD.

The TERT-CLPTM1L lung cancer susceptibility variant associates with higher DNA adduct formation in the lung

Wed, 29 Jul 2009 18:32:35 PDT

Genome-wide association studies have provided evidence that common variation at 5p15.33 [telomerase reverse transcriptase (TERT)-cleft lip and palate transmembrane 1-like (CLPTM1L)], 6p21.33 and 15q25.1 (CHRNA5-CHRNA3) influences lung cancer risk and cancer types with strong environmental risk factors. To independently validate these associations, we compared 5p15.33 (rs402710, rs401681), 6p21.33 (rs4324798) and 15q25.1 (rs1051730, rs16969968 and rs8034191) genotypes in 365 non-small cell lung cancer cases and 440 controls. Consistent with published data, variant genotypes of 5p15 (rs402710), 6p21 and 15q25 showed dose-dependent associations with lung cancer risk. To examine if variants influence the impact of environmental risk factors on lung carcinogenesis, we studied the relationship between genotype and levels of bulky aromatic/hydrophobic DNA adducts in lung tissue adjacent to tumor from 204 lung cancer cases. The risk allele of rs402710 (TERT-CLPTM1L locus) was associated with significantly higher levels of bulky aromatic/hydrophobic DNA adducts (P = 0.02). These data demonstrate a potential association between the TERT-CLPTM1L variant and levels of bulky DNA adducts measured by ³²P-postlabeling and hence a basis for susceptibility to the development of lung cancer.

Association of chromosome 8q variants with prostate cancer risk in Caucasian and Hispanic men

Wed, 29 Jul 2009 18:32:35 PDT

Genotyping of a 615 kb region within 8q24 with 49 haplotype-tagged single-nucleotide polymorphisms (SNPs) in 2109 samples (797 cases and 1312 controls) of two ethnic/racial groups found SNPs that are significantly associated with the risk for prostate cancer (PCa). The highest significance in Caucasian men was found for rs6983267; the AA genotype reduced the risk for PCa [odds ratio (OR) = 0.48, 95% confidence interval (CI) = 0.35–0.65, P = 2.74 x 10^–6]. This SNP also had a significant independent effect from other SNPs in the region in this group. In Hispanic men, rs7837328 and rs921146 showed independent effects (OR = 2.55, 95% CI = 1.51–4.31, P = 4.33 x 10^–4, OR = 2.09, 95% CI = 1.40–3.12, P = 3.13 x 10^–4, respectively). Significant synergist effects for increasing numbers of high-risk alleles were found in both ethnicities. Haplotype analysis revealed major haplotypes, containing the non-risk alleles, conferred protection against PCa. We found high linkage disequilibrium between significant SNPs within the region and SNPs within the CUB and Sushi Multiple Domains 1 gene (CSMD1), on the short arm of chromosome 8 in both ethnicities. These data suggest that multiple interacting SNPs within 8q24, as well as different regions on chromosome 8 far beyond this 8q24 candidate region, may confer increased risk of PCa. This is the first report to investigate the involvement of 8q24 variants in the susceptibility for PCa in Hispanic men.

Chromosome 9 arm-specific telomere length and breast cancer risk

Zheng, Y.-L., Loffredo, C. A., Shields, P. G., Selim, S. M. — Wed, 29 Jul 2009 18:32:35 PDT

Background: Telomere dysfunction is involved in the development of breast cancer and very short telomeres are frequent genetic alterations in breast tumors. However, the influence of telomere lengths of specific chromosomal arms on the breast cancer risk is unknown. Methods: We conducted a case–control study of breast cancer to examine the associations of the telomere length on chromosome 9 short arms (9p) and long arms (9q) with risk of breast cancer. Chromosome 9 arm-specific telomere lengths were measured by quantitative fluorescent in situ hybridization using cultured blood lymphocytes. Results: Telomere length on chromosome 9p was significantly shorter in breast cancer patients than in control subjects (P < 0.001). Using the 50th percentile value in controls as a cut point, women who have short 9p telomeres had an increased risk of breast cancer [adjusted odds ratio (OR) = 2.6; 95% confidence interval (CI) = 1.5–4.3]. When the 9p telomere length was divided into quartiles, a significant inverse dose–response relationship between 9p telomere length and breast cancer risk was observed (P_trend < 0.001), with a quartile ORs of 3.0 (95% CI = 1.2–7.5), 3.9 (95% CI = 1.6–9.5) and 6.6 (95% CI = 2.8–15.9) for third, second and first quartile, respectively, when compared with women in the fourth quartile. Conclusions: Short telomere length on chromosome 9p is strongly associated with the risk of breast cancer. If confirmed by future studies, chromosome 9p telomere length has the potential to be incorporated into the current prediction models to significantly enhance breast cancer risk prediction.

Epigenetic therapy using the histone deacetylase inhibitor for increasing therapeutic gain in oral cancer: prevention of radiation-induced oral mucositis and inhibition of chemical-induced oral carcinogenesis

Chung, Y.-L., Lee, M.-Y., Pui, N. N.M. — Wed, 29 Jul 2009 18:32:35 PDT

In addition to genetic changes, epigenetic aberrations also play important roles in radiation- and chemical-induced disorders and carcinogenesis. The present study investigated whether epigenetic therapy with a histone deacetylase (HDAC) inhibitor has dual benefits for radiation-induced oral mucositis and chemical-induced oral carcinogenesis, which should be treated at the same time. The HDAC inhibitor phenylbutyrate was first tested to determine if it influences DNA damage repair and survival in irradiated normal cells in vitro by investigating the patterns and dynamics of phospho-H2AX foci, Rad51 foci and phospho-H2AX/Rad51 colocalization and using the comet and clonogenic assays. Oral mucositis or carcinogenesis was induced in hamsters using radiation or 7,12-dimethylbenz[a]anthracene (DMBA) irritation to the cheek pouch. The ability of phenylbutyrate formed in proper carriers to prevent radiation-induced oral mucositis and inhibit chemical-induced oral carcinogenesis was assessed. The treated or untreated irradiated or DMBA-irritated oral tissues or mucosal epithelia were subjected to the studies of histology, immunohistochemistry, gene expression, comet assay, HDAC activity or oxidative stress. We found that phenylbutyrate promoted DNA repair and survival in normal cells after radiation. Compared with blank or vehicle-treated hamsters, the irradiated mucosa treated with phenylbutyrate had significantly lower oxidative stress and tumor necrosis factor- expression and less severe oral mucositis of a shorter duration. A reduction of the oral tumor incidence, burden and progression by phenylbutyrate correlated with the suppression of oncomiRs and Rad51 overexpression, the upregulation of differentiation markers and the decrease of intracellular HDAC activity and oxidative stress during DMBA-induced oral carcinogenesis. Thus, epigenetic therapy using the HDAC inhibitor as an adjuvant to radiotherapy for chemical-induced oral cancer may provide a promising strategy combining the prevention of radiation-induced oral mucositis and the inhibition of oral carcinogenesis.

Prenatal N-acetylcysteine prevents cigarette smoke-induced lung cancer in neonatal mice

Balansky, R., Ganchev, G., Iltcheva, M., Steele, V. E., De Flora, S. — Wed, 29 Jul 2009 18:32:35 PDT

Certain adult diseases may have their origin early in life, and perinatal exposures may contribute to cancers both during childhood and later in life. We recently demonstrated that mainstream cigarette smoke (MCS) induces a potent carcinogenic response in mice when exposure starts soon after birth. We also showed that the antioxidant N-acetylcysteine (NAC) prevents the extensive nucleotide and gene expression alterations that occur ‘physiologically’ at birth in mouse lung. The present study was designed to evaluate whether administration of NAC during pregnancy may affect the yield of tumors in mice exposed to MCS, starting after birth and continuing for 120 days. The results obtained showed that 210 days after birth, one adenoma only was detectable in sham-exposed mice. In contrast, as much as the 61.1% (33/54) of MCS-exposed mice born from untreated dams had lung tumors, including both benign tumors and bronchoalveolar carcinomas. Treatment with NAC during pregnancy strikingly inhibited the formation of benign lung tumors and totally prevented occurrence of carcinomas. In addition, prenatal NAC inhibited the MCS-induced hyperplasia of the urinary bladder epithelium. These findings demonstrate for the first time that treatment during pregnancy with an antioxidant chemopreventive agent can affect the induction of tumors consequent to exposure to a carcinogen after birth.

Effect of processed and red meat on endogenous nitrosation and DNA damage

Wed, 29 Jul 2009 18:32:35 PDT

Haem in red meat (RM) stimulates the endogenous production of mutagenic nitroso compounds (NOC). Processed (nitrite-preserved red) meat additionally contains high concentrations of preformed NOC. In two studies, of a fresh RM versus a vegetarian (VEG) diet (six males and six females) and of a nitrite-preserved red meat (PM) versus a VEG diet (5 males and 11 females), we investigated whether processing of meat might increase colorectal cancer risk by stimulating nitrosation and DNA damage. Meat diets contained 420 g (males) or 366 g (females) meat/per day. Faecal homogenates from day 10 onwards were analysed for haem and NOC and associated supernatants for genotoxicity. Means are adjusted for differences in male to female ratios between studies. Faecal NOC concentrations on VEG diets were low (2.6 and 3.5 mmol/g) but significantly higher on meat diets (PM 175 ± 19 nmol/g versus RM 185 ± 22 nmol/g; P = 0.75). The RM diet resulted in a larger proportion of nitrosyl iron (RM 78% versus PM 54%; P < 0.0001) and less nitrosothiols (RM 12% versus PM 19%; P < 0.01) and other NOC (RM 10% versus PM 27%; P < 0.0001). There was no statistically significant difference in DNA breaks induced by faecal water (FW) following PM and RM diets (P = 0.80). However, PM resulted in higher levels of oxidized pyrimidines (P < 0.05). Surprisingly, VEG diets resulted in significantly more FW-induced DNA strand breaks than the meat diets (P < 0.05), which needs to be clarified in further studies. Meats cured with nitrite have the same effect as fresh RM on endogenous nitrosation but show increased FW-induced oxidative DNA damage.

IFN-{gamma}-dependent type 1 immunity is crucial for immunosurveillance against squamous cell carcinoma in a novel mouse carcinogenesis model

Wed, 29 Jul 2009 18:32:35 PDT

3-Methylcholanthrene (MCA)-induced sarcomas have been used as conventional tools for investigating immunosurveillance against tumor development. However, MCA-induced sarcoma is not always an ideal model for the study of the human cancer system because carcinomas and not sarcomas are the dominant types of human cancers. To resolve this problem, we established a novel and simple method to induce mouse squamous cell carcinomas (SCCs). As well known, the subcutaneous injection of MCA caused the formation of sarcomas at 100% incidence. However, we here first succeeded at inducing SCC at 60% of incidence within 2 months by a single intra-dermal injection of MCA. Using this primary SCC model, we demonstrated the critical role of interferon (IFN)--dependent type 1 immunity in immunosurveillance against SCC from the following results: (i) The incidence of SCC was accelerated in IFN--deficient mice compared with that in wild-type mice; (ii) In vivo injection of CpG-oligodeoxynucleotides (CpG-ODN) caused a marked reduction in the incidence of SCC in parallel with the activation of type 1-dependent antitumor immunity and (iii) The antitumor activity of CpG-ODN was significantly decreased in IFN--deficient mice. Thus, our established MCA-induced mouse SCC model could be a powerful tool for evaluating immunosurveillance mechanisms during the development of SCC and might result in a novel strategy to address immunosurveillance mechanisms of human cancer.

{alpha}-Keto acid metabolites of organoselenium compounds inhibit histone deacetylase activity in human colon cancer cells

Nian, H., Bisson, W. H., Dashwood, W.-M., Pinto, J. T., Dashwood, R. H. — Wed, 29 Jul 2009 18:32:35 PDT

Methylselenocysteine (MSC) and selenomethionine (SM) are two organoselenium compounds receiving interest for their potential anticancer properties. These compounds can be converted to β-methylselenopyruvate (MSP) and -keto--methylselenobutyrate (KMSB), -keto acid metabolites that share structural features with the histone deacetylase (HDAC) inhibitor butyrate. We tested the organoselenium compounds in an in vitro assay with human HDAC1 and HDAC8; whereas SM and MSC had little or no activity up to 2 mM, MSP and KMSB caused dose-dependent inhibition of HDAC activity. Subsequent experiments identified MSP as a competitive inhibitor of HDAC8, and computational modeling supported a mechanism involving reversible interaction with the active site zinc atom. In human colon cancer cells, acetylated histone H3 levels were increased during the period 0.5–48 h after treatment with MSP and KMSB, and there was dose-dependent inhibition of HDAC activity. The proportion of cells occupying G₂/M of the cell cycle was increased at 10–50 µM MSP and KMSB, and apoptosis was induced, as evidenced by morphological changes, Annexin V staining and increased cleaved caspase-3, -6, -7, -9 and poly(adenosine diphosphate-ribose)polymerase. P21WAF1, a well-established target gene of clinically used HDAC inhibitors, was increased in MSP- and KMSB-treated colon cancer cells at both the messenger RNA and protein level, and there was enhanced P21WAF1 promoter activity. These studies confirm that in addition to targeting redox-sensitive signaling molecules, -keto acid metabolites of organoselenium compounds alter HDAC activity and histone acetylation status in colon cancer cells, as recently observed in human prostate cancer cells.

Phospholipase C{varepsilon} promotes intestinal tumorigenesis of ApcMin/+ mice through augmentation of inflammation and angiogenesis

Li, M., Edamatsu, H., Kitazawa, R., Kitazawa, S., Kataoka, T. — Wed, 29 Jul 2009 18:32:35 PDT

Apc^Min/+ mice, carrying an inactivated allele of the adenomatous polyposis coli gene, are widely used as an animal model for human colorectal tumorigenesis, where tumor environment, such as inflammation, is known to play a critical role in tumor progression. We previously demonstrated that phospholipase C (PLC), an effector of Ras and Rap small GTPases, plays a crucial role in two-stage skin chemical carcinogenesis using 12-O-tetradecanoyl-phorbor-13-acetate (TPA) as a promoter through augmentation of TPA-induced inflammation. Here, we show that Apc^Min/+ mice lacking PLC (PLC^–/–) exhibit marked resistance to spontaneous intestinal tumorigenesis compared with those with the PLC^+/+ background. Time course of the development of tumors, which are histopathologically classified into low- and high-grade adenomas with increasing dysplasia and size, and adenocarcinomas indicates that not only the low-grade adenoma formation but also the progression to high-grade adenoma are suppressed in PLC^–/–;Apc^Min/+ mice. Low-grade adenomas of PLC^–/–;Apc^Min/+ mice exhibit accelerated apoptosis and reduced cellular proliferation. They also show marked attenuation of tumor angiogenesis and reduction in expression of vascular endothelial growth factor. In contrast, high-grade adenomas of PLC^–/–;Apc^Min/+ mice exhibit marked attenuation of tumor-associated inflammation without significant differences in apoptosis and proliferation. These results suggest that PLC plays crucial roles in intestinal tumorigenesis through two distinct mechanisms, augmentation of angiogenesis and inflammation, depending on the tumor stage.

Increased PEA3/E1AF and decreased Net/Elk-3, both ETS proteins, characterize human NSCLC progression and regulate caveolin-1 transcription in Calu-1 and NCI-H23 NSCLC cell lines

Wed, 29 Jul 2009 18:32:36 PDT

Caveolin-1 protein has been called a ‘conditional tumor suppressor’ because it can either suppress or enhance tumor progression depending on cellular context. Caveolin-1 levels are dynamic in non-small-cell lung cancer, with increased levels in metastatic tumor cells. We have shown previously that transactivation of an erythroblastosis virus-transforming sequence (ETS) cis-element enhances caveolin-1 expression in a murine lung epithelial cell line. Based on high sequence homology between the murine and human caveolin-1 promoters, we proposed that ETS proteins might regulate caveolin-1 expression in human lung tumorigenesis. We confirm that caveolin-1 is not detected in well-differentiated primary lung tumors. Polyoma virus enhancer activator 3 (PEA3), a pro-metastatic ETS protein in breast cancer, is expressed at low levels in well-differentiated tumors and high levels in poorly differentiated tumors. Conversely, Net, a known ETS repressor, is expressed at high levels in the nucleus of well-differentiated primary tumor cells. In tumor cells in metastatic lymph node sites, caveolin-1 and PEA3 are highly expressed, whereas Net is now expressed in the cytoplasm. We studied transcriptional regulation of caveolin-1 in two human lung cancer cell lines, Calu-1 (high caveolin-1 expressing) and NCI-H23 (low caveolin-1 expressing). Chromatin immunoprecipitation-binding assays and small interfering RNA experiments show that PEA3 is a transcriptional activator in Calu-1 cells and that Net is a transcriptional repressor in NCI-H23 cells. These results suggest that Net may suppress caveolin-1 transcription in primary lung tumors and that PEA3 may activate caveolin-1 transcription in metastatic lymph nodes.

Decoy receptor 3, upregulated by Epstein-Barr virus latent membrane protein 1, enhances nasopharyngeal carcinoma cell migration and invasion

Ho, C.-H., Chen, C.-L., Li, W.-Y., Chen, C.-J. — Wed, 29 Jul 2009 18:32:36 PDT

Decoy receptor 3 (DcR3), a member of tumor necrosis factor receptor superfamily, has been implicated in tumorigenesis through its abilities to modulate immune responses and induce angiogenesis. Epstein-Barr virus (EBV), a ubiquitous -herpesvirus, is associated with malignancies including nasopharyngeal carcinoma (NPC). Previous studies show that DcR3 is overexpressed in EBV-positive lymphomas and Rta, an EBV transcription activator, can upregulate DcR3 in Burkitt lymphoma cell lines. However, DcR3 expression has not been demonstrated in EBV-associated NPC nor have there been any EBV latent genes linked to DcR3 upregulation. Here, we showed DcR3 was overexpressed in NPC. Higher DcR3 expression score and DcR3-positive rate were found in metastatic NPC than in primary NPC tissues, suggesting DcR3 may enhance cell metastatic potential. This hypothesis is supported by our observation that NPC HONE-1 cells overexpressing DcR3 exhibited significant higher migration and invasion abilities in vitro. We found besides Rta, EBV latent membrane protein (LMP) 1 can upregulate DcR3 via nuclear factor-kappaB and phosphatidylinositol 3-kinase-signaling events. Approximate 75% of LMP1-positive NPC tissues overexpressed DcR3, suggesting LMP1 may enhance DcR3 expression in vivo. Data herein suggested that increasing DcR3 expression by LMP1 not only helps EBV-associated cancer cells gain survival advantage by preventing host immune detection but also increases the chance of cancer metastasis by enhancing cell migration and invasion. All these DcR3-mediated events facilitate normal cells to gain cancer hallmarks.

Overexpression of MUC15 activates extracellular signal-regulated kinase 1/2 and promotes the oncogenic potential of human colon cancer cells

Wed, 29 Jul 2009 18:32:36 PDT

Mucins play a key role in tumorigenesis. MUC15 is a membrane-bound mucin and the MUC15 messenger RNA (mRNA) has been detected in various organs. However, its role in tumor malignancy is still unclear. This study was to investigate the MUC15 expression in colorectal tumors and the role of MUC15 in colon cancer cells. We found that the mRNA expression of MUC15 was significantly higher in 70.8% (51/72) of colorectal tumors compared with their normal counterparts by real-time reverse transcription–polymerase chain reaction. Immunohistochemistry showed that MUC15 expression was increased in 82.6% (43/52) of colorectal tumors. MUC15 overexpression in HCT116 cells enhanced cell proliferation, cell–extracellular matrix adhesion, colony-forming ability and invasion. Furthermore, these effects were significantly reversed by knockdown of MUC15 with short-hairpin RNA. In nude mice models, MUC15 overexpression significantly (P < 0.01) enhanced tumor growth. In addition, treatment of PD98059 significantly (P < 0.01) inhibited MUC15-enhanced invasion, suggesting that the invasion induced by MUC15 in HCT116 cells was primarily mediated through activation of extracellular signal-regulated kinase 1/2. In conclusion, these results suggest that MUC15 is upregulated in colorectal tumors and its expression enhances the oncogenic potential of colon cancer cells.

Snail2 cooperates with Snail1 in the repression of vitamin D receptor in colon cancer

Wed, 29 Jul 2009 18:32:36 PDT

Vitamin D receptor (VDR) mediates the antitumoral action of the active vitamin D metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). VDR expression is lost during colon cancer progression causing unresponsiveness to 1,25(OH)₂D₃ and its analogs. Previously, Snail1, an inducer of epithelial-to-mesenchymal transition (EMT), was reported to inhibit VDR expression. Here, we show that Snail2/Slug, but not other EMT inducers such as Zeb1, Zeb2, E47 or Twist1, represses VDR gene promoter. Moreover, Snail2 and Snail1 show additive repressing effect on VDR promoter. Snail2 inhibits VDR RNA and protein and blocks the induction of E-cadherin and an adhesive phenotype by 1,25(OH)₂D₃. Snail2 reduces the ligand-induced VDR transcriptional activation of a consensus response element and of the CYP24 promoter. Concordantly, Snail2 inhibits the induction of CYP24 RNA and p21^CIP1, filamin A and vinculin proteins and the repression of c-MYC by 1,25(OH)₂D₃. Additionally, Snail2 abrogates β-catenin nuclear export and the antagonism of the transcriptional activity of β-catenin–T-cell factor complexes by 1,25(OH)₂D₃. SNAI2 expression is upregulated in 58% of colorectal tumors and correlates inversely with that of VDR. However, VDR downregulation is higher in tumors coexpressing SNAI2 and SNAI1 than in those expressing only one of these genes. Together, these data indicate that Snail2 and Snail1 cooperate for VDR repression in colon cancer.