Carcinogenesis - current issue

Backmatter

Mon, 05 Oct 2009 17:18:02 PDT

Frontmatter

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.