Carcinogenesis - recent issues

Frontmatter

2009-06-30

Backmatter

2009-06-30

Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability

Colotta, F., Allavena, P., Sica, A., Garlanda, C., Mantovani, A. — 2009-06-30

Inflammatory conditions in selected organs increase the risk of cancer. An inflammatory component is present also in the microenvironment of tumors that are not epidemiologically related to inflammation. Recent studies have begun to unravel molecular pathways linking inflammation and cancer. In the tumor microenvironment, smoldering inflammation contributes to proliferation and survival of malignant cells, angiogenesis, metastasis, subversion of adaptive immunity, reduced response to hormones and chemotherapeutic agents. Recent data suggest that an additional mechanism involved in cancer-related inflammation (CRI) is induction of genetic instability by inflammatory mediators, leading to accumulation of random genetic alterations in cancer cells. In a seminal contribution, Hanahan and Weinberg [(2000) Cell, 100, 57–70] identified the six hallmarks of cancer. We surmise that CRI represents the seventh hallmark.

Loss of annexin A1 disrupts normal prostate glandular structure by inducing autocrine IL-6 signaling

Inokuchi, J., Lau, A., Tyson, D. R., Ornstein, D. K. — 2009-06-30

Annexin A1 (ANXA1) expression is commonly reduced in premalignant lesions and prostate cancer, but a causal relationship of ANAX1 loss with carcinogenesis has not been established. ANXA1 levels have been shown to inversely correlate with interleukin 6 (IL-6) expression in other cell types and IL-6 has been suggested to enhance prostate cancer initiation and promotion. To investigate whether loss of ANXA1 may contribute to prostate carcinogenesis, ANXA1 expression was reduced using RNA interference in non-tumorigenic human prostatic epithelial cells (RWPE-1/rA1). No effect on morphology, apoptosis, migration or anchorage-dependent or -independent growth was detected. However, IL-6 mRNA and secreted protein levels were elevated in RWPE-1/rA1 cells. In addition, re-expression of ANXA1 in these cells suppressed IL-6 secretion, and altering ANXA1 levels in prostate cancer cells had similar effects on IL-6. The effects of ANXA1 loss and increased IL-6 expression on prostate epithelium were examined using an assay of acinar morphogenesis in vitro. Acini formed by RWPE-1/rA1 cells had delayed luminal clearing and larger mean diameters than control cells. The RWPE-1/rA1 phenotype was recapitulated by treating control cells with recombinant IL-6 and was reversed in RWPE-1/rA1 cells by blocking IL-6 bioactivity. Taken together, these data support a direct role for decreased ANXA1 expression in prostate carcinogenesis and enhancing tumor aggressiveness via the upregulation of IL-6 expression and activity.

c-Met activation in medulloblastoma induces tissue factor expression and activity: effects on cell migration

2009-06-30

Met, the receptor for hepatocyte growth factor (HGF), is a receptor tyrosine kinase that has recently emerged as an important contributor to human neoplasia. In physiological and pathological conditions, Met triggers various cellular functions related to cell proliferation, cell migration and the inhibition of apoptosis, and also regulates a genetic program leading to coagulation. Since medulloblastomas (MBs) express high levels of tissue factor (TF), the main initiator of blood coagulation, we therefore examined the link between Met and TF expression in these pediatric tumors. We observed that stimulation of the MB cell line DAOY with HGF led to a marked increase of TF expression and procoagulant activity, in agreement with analysis of clinical MB tumor specimens, in which tumors expressing high levels of Met also showed high levels of TF. The HGF-dependent increase in TF expression and activity required Src family kinases and led to the translocation of TF to actin-rich structures at the cell periphery, suggesting a role of the protein in cell migration. Accordingly, addition of physiological concentrations of the TF activator factor VIIa (FVII) to HGF-stimulated DAOY cells promoted a marked increase in the migratory potential of these cells. Overall, these results suggest that HGF-induced activation of the Met receptor results in TF expression by MB cells and that this event probably contribute to tumor proliferation by enabling the formation of a provisional fibrin matrix. In addition, TF-mediated non-hemostatic functions, such as migration toward FVIIa, may also play a central role in MB aggressiveness.

Combined inhibition of MET and EGFR suppresses proliferation of malignant mesothelioma cells

2009-06-30

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm associated with asbestos exposure. Although expression and activation of receptor tyrosine kinases (RTKs), including MET, have been reported in most MPM, specific RTK inhibitors showed less than the expected response in MPM cells. To determine whether the lack of response of MET inhibitors was due to cooperation with other RTKs, we determined activation status of MET and other RTKs, including epidermal growth factor receptor (EGFR) family of 20 MPM cell lines, and tested whether dual RTK inhibition is an effective therapeutic strategy. We detected MET upregulation and phosphorylation (thus indicating activation) in 14 (70%) and 13 (65%) cell lines, but treatment with MET-specific inhibitors showed weak or modest effect of suppression in most of the cell lines. Phospho-RTK array analysis revealed that MET was simultaneously activated with other RTKs, including EGFR, ErbB2, ErbB3 and platelet-derived growth factor receptor-β. Combination of MET and EGFR inhibitors triggered stronger inhibition on cell proliferation and invasion of MPM cells than that of each in vitro. These results indicated that coactivation of RTKs was essential in mesothelioma cell proliferation and/or survival, thus suggesting that simultaneous inhibition of RTKs may be a more effective strategy for the development of molecular target therapy for MPM.

Peroxiredoxin I contributes to TRAIL resistance through suppression of redox-sensitive caspase activation in human hepatoma cells

2009-06-30

Reactive oxygen species (ROS) have been implicated in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance of many cancers. We evaluated the role of peroxiredoxin (Prx) I in TRAIL resistance governed by coupling of nicotinamide adenosine dinucleotide phosphate oxidase (Nox)-derived ROS signaling with the p38 mitogen-activated protein kinase (MAPK)/caspase-signaling cascade in liver cancer cells. Upregulated Prx I expression was found in neoplastic regions of human patient liver, and Prx I knockdown resulted in accelerated TRAIL-induced cell death in SK-Hep-1 human hepatoma cells. The TRAIL cytotoxicity by Prx I knockdown was dependent on activation of caspase-8/3 cascades, which was ablated by addition of inhibitors for p38 MAPK, ROS or Nox, suggesting the association with Nox-driven redox signaling. Furthermore, we found that Nox4 was constitutively expressed in both SK-Hep-1 cells and tumor regions of patient livers, knockdown of Nox4 expression could alleviate ROS generation and TRAIL-mediated cytotoxicity. In accordance with previous findings, increased activation of both p38 MAPK and caspase cascades by Prx I knockdown was inhibited by either Nox4 knockdown or SB203580 addition. Collectively, these data suggest that Prx I functions to block propagation of Nox-derived ROS signaling to the p38 MAPK/caspase/cell death cascade during TRAIL treatment and also provides a molecular mechanism by which Prx I contributes to TRAIL resistance in liver cancers.

The isatin-Schiff base copper(II) complex Cu(isaepy)2 acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

2009-06-30

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N']copper(II) [Cu(isaepy)₂] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)₂ to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)₂ increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)₂ behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)₂ in apoptosis is confirmed by experiments carried out with ⁰ cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)₂. Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate:ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)₂-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)₂ behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.

{alpha}-Tocopheryl succinate and derivatives mediate the transcriptional repression of androgen receptor in prostate cancer cells by targeting the PP2A-JNK-Sp1-signaling axis

Huang, P.-H., Wang, D., Chuang, H.-C., Wei, S., Kulp, S. K., Chen, C.-S. — 2009-06-30

As part of our effort to understand the mechanism underlying -tocopheryl succinate [vitamin E succinate (VES)]-mediated antitumor effects, we investigated the signaling pathway by which VES suppresses androgen receptor (AR) expression in prostate cancer cells. VES and, to a greater extent, its truncated derivative TS-1 mediated transcriptional repression of AR in prostate cancer cells but not in normal prostate epithelial cells; a finding that underscores the differential susceptibility of normal versus malignant cells to the antiproliferative effect of these agents. This AR repression was attributable to the ability of VES and TS-1 to facilitate the proteasomal degradation of the transcription factor Sp1. This mechanistic link was corroborated by the finding that proteasome inhibitors or ectopic expression of Sp1 protected cells against drug-induced AR ablation. Furthermore, evidence suggests that the destabilization of Sp1 by VES and TS-1 resulted from the inactivation of Jun N-terminal kinases (JNKs) as a consequence of increased phosphatase activity of protein phosphatase 2A (PP2A). Stable transfection of LNCaP cells with the dominant-negative JNK1 plasmid mimicked drug-induced Sp1 repression, whereas constitutive activation of JNK kinase activity or inhibition of PP2A activity by okadaic acid protected Sp1 from VES- and TS-1-induced degradation. From a mechanistic perspective, the ability of VES and TS-1 to activate PP2A activity underscores their broad spectrum of effects on multiple signaling mechanisms, including those mediated by Akt, mitogen-activated protein kinases, nuclear factor kappaB, Sp1 and AR. This pleiotropic effect in conjunction with low toxicity suggests the translational potential for developing TS-1 into potent PP2A-activating agents for cancer therapy.

Concomitant promoter methylation of multiple genes in lung adenocarcinomas from current, former and never smokers

2009-06-30

Aberrant promoter hypermethylation is one of the major mechanisms in carcinogenesis and some critical growth regulatory genes have shown commonality in methylation across solid tumors. Twenty-six genes, 14 identified through methylation in colon and breast cancers, were evaluated using primary lung adenocarcinomas (n = 175) from current, former and never smokers. Tumor specificity of methylation was validated through comparison of 14 lung cancer cell lines to normal human bronchial epithelial cells derived from bronchoscopy of 20 cancer-free smokers. Twenty-five genes were methylated in 11–81% of primary tumors. Prevalence for methylation of TNFRSF10C, BHLHB5 and BOLL was significantly higher in adenocarcinomas from never smokers than smokers. The relation between methylation of individual genes was examined using pairwise comparisons. A significant association was seen between 138 (42%) of the possible 325 pairwise comparisons. Most notably, methylation of MMP2, BHLHB4 or p16 was significantly associated with methylation of 16–19 other genes, thus predicting for a widespread methylation phenotype. Kaplan–Meier log-rank test and proportional hazard models identified a significant association between methylation of SULF2 (a pro-growth, -angiogenesis and -migration gene) and better patient survival (hazard ratio = 0.23). These results demonstrate a high degree of commonality for targeted silencing of genes between lung and other solid tumors and suggest that promoter hypermethylation in cancer is a highly co-ordinated event.

Overexpression of SMYD2 relates to tumor cell proliferation and malignant outcome of esophageal squamous cell carcinoma

2009-06-30

Although we have identified two putative targets, ATF3 and CENPF, for a frequently gained/amplified region around 1q32–q41 in esophageal squamous cell carcinoma (ESCC), it is possible that other amplification targets remain to be identified. In this study, we tested whether SET and MYND domain-containing protein 2 (SMYD2), located between those two genes and encoding a lysine methyltransferase for histone H3K36 and p53K370 that regulates transcription and inhibits transactivation activity, respectively, acts as a cancer-promoting gene through activation/overexpression in ESCC. Frequent overexpression of SMYD2 messenger RNA and protein was observed in KYSE150 cells with remarkable amplification at 1q32–41.1 and other ESCC cell lines (11/43 lines, 25.6%). Overexpression of SMYD2 protein was frequently detected in primary tumor samples of ESCC (117/153 cases, 76.5%) as well and significantly correlated with gender, venous invasion, the pT category in the tumor–lymph node–metastases classification and status of recurrence. Patients with SMYD2-overexpressing tumors had a worse overall rate of survival than those with non-expressing tumors, and SMYD2 positivity was independently associated with a worse outcome in the multivariate analysis. Knockdown of SMYD2 expression inhibited and ectopic overexpression of SMYD2 promoted the proliferation of ESCC cells in a TP53 mutation-independent but SMYD2 expression-dependent manner. These findings suggest that SMYD2 plays an important role in tumor cell proliferation through its activation/overexpression and highlight its usefulness as a prognosticator and potential therapeutic target in ESCC.

Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma

2009-06-30

The human hMYH and NEIL1 genes encode DNA glycosylases involved in repair of oxidative base damage and mutations in these genes are associated with certain cancers. Primary sclerosing cholangitis (PSC), a chronic cholestatic liver disease characterized by inflammatory destruction of the biliary tree, is often complicated by the development of cholangiocarcinoma (CCA). Here, we aimed to investigate the influence of genetic variations in the hMYH and NEIL1 genes on risk of CCA in PSC patients. The hMYH and NEIL1 gene loci in addition to the DNA repair genes hOGG1, NTHL1 and NUDT1 were analyzed in 66 PSC patients (37 with CCA and 29 without cancer) by complete genomic sequencing of exons and adjacent intronic regions. Several single-nucleotide polymorphisms and mutations were identified and severe impairment of protein function was observed for three non-synonymous variants. The NEIL1 G83D mutant was dysfunctional for the major oxidation products 7,8-dihydro-8-oxoguanine (8oxoG), thymine glycol and dihydrothymine in duplex DNA, and the ability to perform -elimination at abasic sites was significantly reduced. The hMYH R260Q mutant had severe defect in adenine DNA glycosylase activity, whereas hMYH H434D could excise adenines from A:8oxoG pairs but not from A:G mispairs. We found no overall associations between the 18 identified variants and susceptibility to CCA in PSC patients; however, the impaired variants may be of significance for carcinogenesis in general. Our findings demonstrate the importance of complete resequencing of selected candidate genes in order to identify rare genetic variants and their possible contribution to individual susceptibility to cancer development.

EGFR pathway polymorphisms and bladder cancer susceptibility and prognosis

2009-06-30

The epidermal growth factor receptor (EGFR) pathway has recently been appreciated as a central mediator of tumorigenesis and an important drug target; however, the influence of genetic variation in this pathway on bladder cancer is not understood. Pathway activation leads to cell proliferation, angiogenesis and is antiapoptotic. We sought to test the hypothesis that bladder cancer susceptibility and survival are modified by inherited variations in the sequence of the EGFR and its pathway members. We tested associations using a population-based study of 857 bladder cancer cases and 1191 controls from New Hampshire. Multifactor dimensionality reduction software was used to predict gene–gene interactions. We detected an increased risk of bladder cancer associated with variant genotypes for the single nucleotide polymorphisms EGFR_03 [adjusted odds ratio (OR) 1.7 (95% confidence interval (CI) 1.0–2.8)] and EGFR_05 [adjusted OR 1.5 (95% CI 1.0–2.1)] compared with wild-type. EGFR variants experienced longer survival than those with wild-type alleles [e.g. adjusted hazard ratio EGFR_1808 0.3 (95% CI 0.1–0.9)]. In contrast, the variant form of the ligand, EGF_04, had worse survival [adjusted hazard ratio 1.5 (95% CI 1.0–2.3)] compared with wild-type. Our findings suggest modified bladder cancer risk and survival associated with genetic variation in the EGFR pathway. Understanding these genetic influences on increased bladder cancer susceptibility and survival may help in cancer prevention, drug development and choice of therapeutic regimen.

CYP450 polymorphisms as risk factors for early-onset lung cancer: gender-specific differences

2009-06-30

Cytochrome P450 (CYP) enzymes, involved in metabolism of tobacco carcinogens, are also involved in estrogen metabolism and many are regulated by estrogens. These genes may thus be of relevance to gender-specific differences in lung cancer risk, particularly in early-onset lung cancer, where a high proportion of women is observed. We conducted a case–control study to investigate genetic polymorphisms in cytochromes that might modify the risk of developing early-onset lung cancer. In total, 638 Caucasian patients under the age of 51 with primary lung cancer and 1300 cancer-free control individuals, matched by age and sex, were included in this analysis. Thirteen polymorphisms in the CYP1A1, CYP1B1, CYP2A13, CYP3A4 and CYP3A5 genes were analyzed. No significant association was found for any of the analyzed polymorphisms and lung cancer risk overall. However, among women, a significantly increased risk of early-onset lung cancer was observed for carriers of the minor allele of CYP1B1 SNP rs1056836 [odds ratio (OR) 1.97; 95% confidence interval (CI) 1.32–2.94; P < 0.001]. Also, a non-significant increase in lung cancer risk was observed in the group of women carriers of the minor allele of CYP2A13 SNP rs1709084 (OR 1.64; 95% CI 1.00–2.70; P = 0.05). The effect of these two polymorphisms was shown to be modified by smoking. Haplotype analysis was performed for CYP1B1 and CYP2A13. No differences between cases and controls were observed for both genes (P = 0.63 and P = 0.42 for CYP1B1 and CYP2A13, respectively). Our results suggest that the CYP1B1 and the CYP2A13 genotypes may contribute to individual susceptibility to early-onset lung cancer in women.

Review and meta-analysis on vitamin D receptor polymorphisms and cancer risk

Raimondi, S., Johansson, H., Maisonneuve, P., Gandini, S. — 2009-06-30

It was suggested that vitamin D levels influence cancer development. The vitamin D receptor (VDR) is a crucial mediator for the cellular effects of vitamin D. Results from previous studies on the association of VDR polymorphisms with different cancer types are somewhat contradictory, and the role of VDR in the etiology of cancer is still equivocal. We therefore performed a meta-analysis on the association between the two most studied VDR polymorphisms (FokI and BsmI) and any cancer site. Up to January 2009, we identified 67 independent studies. We used random-effects models to provide summary odds ratio (SOR) for VDR polymorphisms and cancer. We tested homogeneity of effects across studies and publication bias and explored between-study heterogeneity. When comparing FokI ff with FF carriers, we found a significant increase in skin cancer [SOR; 95% confidence intervals (CIs): 1.30; 1.04–1.61] and breast cancer (SOR; 95%CI: 1.14; 1.03–1.27) risk. For the same genotype comparison, we found a significantly higher risk of cancer when we pooled estimates from cancer sites possibly associated with vitamin D levels (prostate, breast, skin, ovary, non-Hodgkin lymphoma and colorectal). A significant reduction in prostate cancer risk was observed for carriers of BsmI Bb compared with bb genotype (SOR; 95%CI: 0.83; 0.69–0.99). In Caucasian populations, both Bb and BB carriers had a significant reduced risk of cancer at any site. In conclusion, this meta-analysis showed that VDR FokI and BsmI polymorphisms might modulate the risk of cancer of breast, skin and prostate and possibly affect cancer risk at any site in Caucasians.

HapMap-based study of the DNA repair gene ERCC2 and lung cancer susceptibility in a Chinese population

Yin, J., Vogel, U., Ma, Y., Qi, R., Wang, H. — 2009-06-30

DNA repair genes have been proposed as candidate cancer susceptibility genes. The excision repair cross-complementing rodent repair deficiency, complementation group 2 (ERCC2)/xeroderma pigmentosum complementary group D (XPD) protein is considered to be a key enzyme in nucleotide excision repair (NER) pathway. To elucidate whether common ERCC2 variants are associated with lung cancer susceptibility, we conducted a case–control study consisting of 339 cases with primary lung cancer and 358 controls matched on age, gender and ethnicity in a Chinese population. Six haplotype tagging single-nucleotide polymorphisms (htSNPs) (rs238403, rs50871, rs3916840, rs238415, rs3916874 and rs1799787) from HapMap database were analyzed, which provide an almost complete coverage of the genetic variations in the ERCC2 gene. Although none of the six htSNPs was individually associated with lung cancer risk, we found that two ERCC2 haplotypes were associated with risk of lung cancer. Haplotype 4 defined by rs238403T-rs50871T-rs3916840C-rs238415C-rs3916874G-rs1799787C and haplotype 7 defined by rs238403C-rs50871G-rs3916840C-rs238415C-rs3916874G-rs1799787C were strongly associated with an increased risk of lung cancer [odds ratio, OR (95% confidence interval, CI) = 2.62 (1.53–4.50), P = 0.0003 for hap4; OR (95% CI) = 3.01 (1.36–6.63), P = 0.004 for hap7]. Furthermore, diplotype analyses also strengthened the significant associations of risk haplotype 4 [OR (95% CI) = 3.56 (2.12–5.87), P < 0.001] or risk haplotype 7 [OR (95% CI) = 3.38 (1.75–6.55), P < 0.001] and lung cancer. Analysis of linkage disequilibrium (LD) also confirmed that considerable LD exists between the pairs of the six htSNPs within ERCC2. These results suggested that the risk subhaplotypes cosegregate with one or more biologically functional polymorphisms. Our results provide evidence to support a role for ERCC2 in lung cancer development in a Chinese population.

A specific interleukin-1B haplotype correlates with high levels of IL1B mRNA in the lung and increased risk of non-small cell lung cancer

2009-06-30

Epidemiological evidence suggests a relationship between chronic inflammation and lung cancer. Inflammation in the lung may be modulated by host genetic factors such as polymorphisms in inflammatory genes. Identification of polymorphisms in inflammatory genes may help understanding interindividual differences in susceptibility to lung cancer. We have investigated single-nucleotide polymorphisms (SNPs) and their haplotypes in the regulatory region of the IL1B gene in association to non-small cell lung cancer (NSCLC) risk. Our previous work showed that two promoter SNPs C-511T and T-31C modulated NSCLC risk. In the present study, we show that G-3893A and G-1464C located in the enhancer region of the IL1B gene may also affect this risk, with odds for developing NSCLC being 0.69 [95% confidence interval (CI), 0.52–0.92] for -3893 A-allele and 0.63 (95% CI, 0.47 – 0.83) for -1464 C-allele. The associations were particularly prominent in patients with TP53 mutations in the tumor. Inference of the haplotype structures showed that -3893 G, -1464 G, -511 C and -31 T formed a specific haplotype (GGCT) with near complete linkage disequilibrium in lung cancer patients but not in controls. Furthermore, the risk haplotype (GGCT) was present in 65% of cases compared with 36% of controls. Quantitative analysis of RNA in normal lung tissue of the patients showed that the risk haplotype was correlated with significantly higher IL1B messenger RNA (mRNA) levels compared with the non-risk haplotype (ACTC). These data suggest that a specific IL1B haplotype associated with increased IL1B gene expression increases the risk of NSCLC.

Tolfenamic acid inhibits esophageal cancer through repression of specificity proteins and c-Met

2009-06-30

The non-steroidal anti-inflammatory drug tolfenamic acid (TA) inhibits proliferation of SEG-1 and BIC-1 esophageal cancer cells with half-maximal growth inhibitory concentration values of 36 and 48 µM, respectively. TA also increased Annexin V staining in both cell lines, indicative of proapoptotic activity. Treatment of SEG-1 and BIC-1 cells with TA for up to 72 h decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and this was accompanied by decreased expression of the well-characterized Sp-regulated genes cyclin D1, vascular endothelial growth factor and survivin. TA also decreased hepatocyte growth factor receptor, (c-Met), a receptor tyrosine kinase that is overexpressed in esophageal cancer cells and tumors and is an important drug target. Knockdown of Sp1, Sp3 and Sp4 by RNA interference in SEG-1 and BIC-1 cells also decreased c-Met expression, demonstrating that c-Met is an Sp-regulated gene in esophageal cancer cells. Sp1 was overexpressed in esophageal cancer cells and tumors and increased Sp1 staining was observed in esophageal tumors from patients. TA (20 mg/kg/day) also decreased tumor growth and weight in athymic nude mice bearing SEG-1 cells as xenografts and this was accompanied by increased apoptosis and decreased Sp1 and c-Met staining in tumors from treated mice. Thus, TA-dependent downregulation of Sp transcription factors and c-Met defines a novel chemotherapeutic approach for treatment of esophageal cancer.

Vitamin C and {alpha}-naphthoflavone prevent estrogen-induced mammary tumors and decrease oxidative stress in female ACI rats

Mense, S. M., Singh, B., Remotti, F., Liu, X., Bhat, H. K. — 2009-06-30

The mechanisms underlying the pathogenesis of estrogen-induced breast carcinogenesis remain unclear. The present study investigated the roles of estrogen metabolism and oxidative stress in estrogen-mediated mammary carcinogenesis in vivo. Female August Copenhagen Irish (ACI) rats were treated with 17β-estradiol (E₂), the antioxidant vitamin C, the estrogen metabolic inhibitor -naphthoflavone (ANF), or cotreated with E₂ + vitamin C or E₂ + ANF for up to 8 months. E₂ (3 mg) was administered as an subcutaneous implant, ANF was given via diet (0.2%) and vitamin C (1%) was added to drinking water. At necropsy, breast tumor incidence in the E₂, E₂ + vitamin C and E₂ + ANF groups was 82, 29 and 0%, respectively. Vitamin C and ANF attenuated E₂-induced alterations in oxidative stress markers in breast tissue, including 8-iso-prostane F₂ formation and changes in the activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase. Quantification of 2-hydroxyestradiol (2-OHE₂) and 4-hydroxyestradiol (4-OHE₂) formation in breast tissue confirmed that ANF inhibited 4-hydroxylation of E₂ and decreased formation of the highly carcinogenic 4-OHE₂. These results demonstrate that antioxidant vitamin C reduces the incidence of estrogen-induced mammary tumors, increases tumor latency and decreases oxidative stress in vivo. Further, our data indicate that ANF completely abrogates breast cancer development in ACI rats. The present study is the first to demonstrate the inhibition of breast carcinogenesis by antioxidant vitamin C or the estrogen metabolic inhibitor ANF in an animal model of estrogen-induced mammary carcinogenesis. Taken together, these results suggest that E₂ metabolism and oxidant stress are critically involved in estrogen-induced breast carcinogenesis.

Linoleic acid metabolite suppresses skin inflammation and tumor promotion in mice: possible roles of programmed cell death 4 induction

2009-06-30

(±)-13-Hydroxy-10-oxo-trans-11-octadecenoic acid (13-HOA) is one of the lipoxygenase metabolites of linoleic acid (LA) from corn germ. Recently, we reported that this metabolite suppressed the expression of lipopolysaccharide-induced proinflammatory genes in murine macrophages by disrupting mitogen-activated protein kinases and Akt pathways. In this study, we investigated the inhibitory effects of 13-HOA on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in ears and skin, as well as tumor promotion in female ICR mice. Pretreatment with 13-HOA (1600 nmol) inhibited ear edema formation by 95% (P < 0.05) in an inflammation test and reduced tumor incidence and the number of tumors per mouse by 40 and 64% (P < 0.05 each), respectively, in a two-stage skin carcinogenesis model. Histological examinations revealed that it decreased epidermal thickness, the number of infiltrated leukocytes and cell proliferation index. Furthermore, 13-HOA (8–40 µM) suppressed TPA-induced anchorage-independent growth of JB6 mouse epidermal cells by 70–100%, whereas LA was virtually inactive. 13-HOA (40 µM) inhibited TPA-induced activator protein-1 transactivation but not extracellular signal-regulated kinase1/2 activation. Interestingly, 13-HOA (40 µM and 1600 nmol in JB6 cells and mouse skin, respectively) induced expression of programmed cell death 4 (Pdcd4), a novel tumor suppressor protein. To our knowledge, this is the first report of a food factor that is able to induce Pdcd4 expression. Collectively, our results indicate that 13-HOA may be a novel anti-inflammatory and antitumor chemopreventive agent with a unique mode of action.

5-Aminosalicylic acid inhibits colitis-associated but not sporadic colorectal neoplasia in a novel conditional Apc mouse model

2009-06-30

Genetic predisposition, life-style habits and inflammatory bowel diseases (IBD)-related colitis are a main risk factor for colorectal cancer (CRC). 5-Aminosalicylic acid (5-ASA, mesalazine) is a mainstay therapy in IBD and believed to reduce the risk for developing CRC. We aimed to determine the ability of 5-ASA enemas to inhibit the development of sporadic and colitis-related neoplasia in mice. FabplCre;Apc^15lox/+ mice, which spontaneously develop sporadic colorectal tumours, were treated at 5 weeks of age with 5-ASA or placebo enemas for 3 weeks and examined for colorectal tumourigenesis at 8 weeks of age. Colitis-related tumour development was investigated in these mice by administration of dextran sodium sulphate, inducing intestinal inflammation and accelerating colorectal tumourigenesis, combined with treatment of 5-ASA or placebo enemas during and/or after colitis induction. 5-ASA significantly reduced colitis-accelerated neoplasia development by 50%, from 19.4 ± 2.7 to 9.4 ± 2.4 (mean tumour numbers ± SEM, P = 0.02), in the distal part of the large intestine covered by the enema. 5-ASA was only effective when given during and/or after the intestinal inflammatory period. 5-ASA did not reduce, however, sporadic neoplasia development in the FabplCre;Apc^15lox/+ mice. 5-ASA tended to reduce proliferation of epithelial cells in the colitis-associated colorectal tumours but not in the sporadic colorectal tumours. In conclusion, 5-ASA medication inhibits the development of colitis-associated tumours in FabplCre;Apc^15lox/+ mice when administered during and/or after the induction of inflammation. 5-ASA does not reduce, however, sporadic tumour development in this mouse model.

Kalopanaxsaponin A inhibits PMA-induced invasion by reducing matrix metalloproteinase-9 via PI3K/Akt- and PKC{delta}-mediated signaling in MCF-7 human breast cancer cells

Park, S. K., Hwang, Y. S., Park, K.-K., Park, H.-J., Seo, J. Y., Chung, W.-Y. — 2009-06-30

Induction of matrix metalloproteinase (MMP)-9 is particularly important for the invasiveness of breast cancers. We investigated the inhibitory effect of kalopanaxsaponin A (KPS-A) on cell invasion and MMP-9 activation in phorbol 12-myristate 13-acetate (PMA)-treated MCF-7 human breast cancer cells. KPS-A inhibited PMA-induced cell proliferation and invasion. PMA-induced cell invasion was blocked in the presence of a primary antibody of MMP-9, and KPS-A suppressed the increased expression and/or secretion of MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1. Using specific inhibitors, we confirmed that PMA-induced cell invasion and MMP-9 expression is primarily regulated by nuclear factor-kappa B (NF-B) activation via phosphatidylinositol 3-kinase (PI3K)/Akt and activator protein-1 (AP-1) activation via extracellular signal-regulated kinase (ERK)1/2. KPS-A decreased PMA-induced transcriptional activation of NF-B and AP-1 and inhibited PMA-induced phosphorylation of ERK1/2 and Akt. Treatment with the protein kinase C (PKC) inhibitor rottlerin caused a marked decrease in PMA-induced MMP-9 secretion and cell invasion, as well as ERK/AP-1 activation, and KPS-A reduced PMA-induced membrane localization of PKC. Furthermore, oral administration of KPS-A led to a substantial decrease in tumor volume and expression of proliferating cell nuclear antigen, MMP-9, TIMP-1 and PKC in mice with MCF-7 breast cancer xenografts in the presence of 17β-estradiol. These results suggest that KPS-A inhibits PMA-induced invasion by reducing MMP-9 activation, mainly via the PI3K/Akt/NF-B and PKC/ERK/AP-1 pathways in MCF-7 cells and blocks tumor growth and MMP-9-mediated invasiveness in mice with breast carcinoma. Therefore, KPS-A may be a promising anti-invasive agent with the advantage of oral dosing.

Pterostilbene inhibited tumor invasion via suppressing multiple signal transduction pathways in human hepatocellular carcinoma cells

Pan, M.-H., Chiou, Y.-S., Chen, W.-J., Wang, J.-M., Badmaev, V., Ho, C.-T. — 2009-06-30

Pterostilbene, a natural dimethylated analog of resveratrol, is known to have diverse pharmacologic activities including anticancer, anti-inflammation, antioxidant, apoptosis, anti-proliferation and analgesic potential. However, the effects of pterostilbene in preventing invasion of cancer cells have not been studied. Here, we report our finding that pterostilbene significantly suppressed 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced invasion, migration and metastasis of human hepatoma cells (HepG₂ cells). Increase in the enzyme activity, protein and messenger RNA levels of matrix metalloproteinase (MMP)-9 were observed in TPA-treated HepG₂ cells, and these were blocked by pterostilbene. In addition, pterostilbene can inhibit TPA-induced expression of vascular endothelial growth factor, epidermal growth factor and epidermal growth factor receptor. Transient transfection experiments also showed that pterostilbene strongly inhibited TPA-stimulated nuclear factor kappa B (NF-B) and activator protein-1 (AP-1)-dependent transcriptional activity in HepG₂ cells. Moreover, pterostilbene can suppress TPA-induced activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, c-Jun N-terminal kinases 1/2 and phosphatidylinositol 3-kinase/Akt and protein kinase C that are upstream of NF-B and AP-1. Significant therapeutic effects were further demonstrated in vivo by treating nude mice with pterostilbene (50 and 250 mg/kg intraperitoneally) after inoculation with HepG₂ cells into the tail vein. Presented data reveal that pterostilbene is a novel, effective, anti-metastatic agent that functions by downregulating MMP-9 gene expression.

Alterations of histone modifications by cobalt compounds

Li, Q., Ke, Q., Costa, M. — 2009-06-30

In the present study, we examined the effects of CoCl₂ on multiple histone modifications at the global level. We found that in both human lung carcinoma A549 cells and human bronchial epithelial Beas-2B cells, exposure to CoCl₂ (≥200 µM) for 24 h increased H3K4me3, H3K9me2, H3K9me3, H3K27me3, H3K36me3, uH2A and uH2B but decreased acetylation at histone H4 (AcH4). Further investigation demonstrated that in A549 cells, the increase in H3K4me3 and H3K27me3 by cobalt ions exposure was probably through enhancing histone methylation processes, as methionine-deficient medium blocked the induction of H3K4me3 and H3K27me3 by cobalt ions, whereas cobalt ions increased H3K9me3 and H3K36me3 by directly inhibiting JMJD2A demethylase activity in vitro, which was probably due to the competition of cobalt ions with iron for binding to the active site of JMJD2A. Furthermore, in vitro ubiquitination and deubiquitination assays revealed that the cobalt-induced histone H2A and H2B ubiquitination is the result of inhibition of deubiquitinating enzyme activity. Microarray data showed that exposed to 200 µM of CoCl₂ for 24 h, A549 cells not only increased but also decreased expression of hundreds of genes involved in different cellular functions, including tumorigenesis. This study is the first to demonstrate that cobalt ions altered epigenetic homeostasis in cells. It also sheds light on the possible mechanisms involved in cobalt-induced alteration of histone modifications, which may lead to altered programs of gene expression and carcinogenesis since cobalt at higher concentrations is a known carcinogen.

Sequestration of E12/E47 and suppression of p27KIP1 play a role in Id2-induced proliferation and tumorigenesis

2009-06-30

Id2 is a member of the helix-loop-helix (HLH) family of transcription regulators known to antagonize basic HLH transcription factors and proteins of the retinoblastoma tumor suppressor family and is implicated in the regulation of proliferation, differentiation, apoptosis and carcinogenesis. To investigate its proposed role in tumorigenesis, Id2 or deletion mutants were re-expressed in Id2^–/– dermal fibroblasts. Ectopic expression of Id2 or mutants containing the central HLH domain increased S-phase cells, cell proliferation in low and normal serum and induced tumorigenesis when grafted or subcutaneously injected into athymic mice. Similar to their downregulation in human tumors, the expression of cyclin-dependent kinase inhibitors p27^KIP1 and p15^INK4b was decreased by Id2; the former by downregulation of its promoter by the Id2 HLH domain-mediated sequestration of E12/E47. Re-expression of p27^KIP1 in Id2-overexpressing cells reverted the hyperproliferative and tumorigenic phenotype, implicating Id2 as an oncogene working through p27^KIP1. These results tie together the previously observed misregulation of Id2 with a novel mechanism for tumorigenesis.

DNA double-strand break repair activities in mammary epithelial cells--influence of endogenous p53 variants

Keimling, M., Wiesmuller, L. — 2009-06-30

Intriguingly, all 10 breast cancer susceptibility genes known today are directly or indirectly related to DNA double-strand break (DSB) repair suggesting a critical role of DSB repair dysfunction in the etiology of this tumor entity. We and others had previously provided evidence indicating that the breast cancer susceptibility gene product p53 controls DSB repair. Experiments with ectopically expressed proteins showed that oncogenic mutants of p53 deregulate homologous recombination (HR) and possibly also non-homologous end joining (NHEJ). Here, we systematically analyzed the role of different p53 variants endogenously expressed in a series of mammary epithelial cell lines. We provide evidence that endogenous wild-type p53 represses HR, particularly between short homologies that strengthens the idea of a quality control mechanism underlying HR regulation. To a lesser extent, p53 also downregulates microhomology-mediated NHEJ and single-strand annealing. Our data also suggest that repression of NHEJ regulation may require the extreme C-terminus, whereas the oligomerization and core domains are involved in HR regulation. We show that depending on the individual mutation, p53 mutants retain more or less partial DSB repair downregulatory activities when compared with loss of p53. All in all, relative effects on distinct DSB repair pathways and discrimination between HR substrates with perfectly versus imperfectly homologous sequences represent good markers for a p53 defect due to a specific mutation. Thus, advanced DSB repair analysis may serve as a novel assay for the functional classification of p53 mutations.

Frontmatter

2009-06-03

Backmatter

2009-06-03

Hedgehog signalling in breast cancer

Kasper, M., Jaks, V., Fiaschi, M., Toftgard, R. — 2009-06-03

Breast cancer is the most common cause of cancer death among women worldwide. In order to improve the treatment of this disease, a more complete understanding of its biological basis is necessary. Since the Hedgehog (Hh) pathway was recently found to be required for growth and propagation of a number of different cancers, we discuss here the possible involvement of this pathway in the normal biology and development of cancer in the mammary gland. The use of mouse mammary cancer models has assisted the process of dissecting the mechanisms behind Hh-driven mammary tumour formation and growth. Based on recent studies, we conclude that the inhibition of Hh signalling in breast tumours may interfere with the maintenance of a putative cancer stem cell compartment and the abnormal stimulation of tumour stroma. Therefore, the components of the Hh signalling cascade may provide a set of drug targets, which could be implemented into novel combinatorial strategies for the treatment of breast cancer.

MicroRNAs and genomic variations: from Proteus tricks to Prometheus gift

Fabbri, M., Valeri, N., Calin, G. A. — 2009-06-03

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory functions. MiRNAs are aberrantly expressed in almost all human cancers, leading to abnormal levels of target genes. Recently, an increasing number of studies have addressed whether genomic variations including germ line or somatic mutations and single-nucleotide polymorphisms can count for miRNA abnormal expression by altering their biogenesis and/or affect the ability of miRNAs to bind to target messenger RNAs. Here, we provide an extensive review of the studies that have investigated variations occurring both in miRNA genes and in target genes and we discuss the possible clinical implications of these findings. Furthermore, we propose that sequence variations in miRNAs or interactor sites located in mRNAs can be involved in cancer predisposition.

Time-point and dosage of gene inactivation determine the tumor spectrum in conditional Ptch knockouts

2009-06-03

Mutations in Patched (PTCH) have been associated with tumors characteristic both for children [medulloblastoma (MB) and rhabdomyosarcoma (RMS)] and for elderly [basal cell carcinoma (BCC)]. The determinants of the variability in tumor onset and histology are unknown. We investigated the effects of the time-point and dosage of Ptch inactivation on tumor spectrum using conditional Ptch-knockout mice. Ptch heterozygosity induced prenatally resulted in the formation of RMS, which was accompanied by the silencing of the remaining wild-type Ptch allele. In contrast, RMS was observed neither after mono- nor biallelic postnatal deletion of Ptch. Postnatal biallelic deletion of Ptch led to BCC precancerous lesions of the gastrointestinal epithelium and mesenteric tumors. Hamartomatous gastrointestinal cystic tumors were induced by monoallelic, but not biallelic Ptch mutations, independently of the time-point of mutation induction. These data suggest that the expressivity of Ptch deficiency is largely determined by the time-point, the gene dose and mode of Ptch inactivation. Furthermore, they point to key differences in the tumorigenic mechanisms underlying adult and childhood tumors. The latter ones are unique among all tumors since their occurrence decreases rather than increases with age. A better understanding of mechanisms underlying this ontological restriction is of potential therapeutic value.

Suppression of NF-{kappa}B activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells

Kim, A., Kim, M.-J., Yang, Y., Kim, J. W., Yeom, Y. I., Lim, J.-S. — 2009-06-03

Downregulation of the N-myc downstream-regulated gene 2 (NDRG2) gene is involved in the progression of aggressive forms of cancer, along with the poor prognosis of cancer patients. In the current study, we examined the effect of NDRG2 expression on the metastatic potential of HT1080 human fibrosarcoma and B16F10 murine melanoma cells in both in vitro and in vivo systems. In gelatin zymography, NDRG2 expression remarkably suppressed the matrix metalloproteinase (MMP)-9 activity and slightly inhibited MMP-2 activity of both cell lines. Tumor migration and invasion in vitro were significantly reduced by NDRG2 expression, and NDRG2 inhibited tumor cell proliferation in an anchorage-independent semisolid agar assay. Specifically, we found that NDRG2 affects invasion through suppression of nuclear factor kappa B (NF-B) activity. In animal experiments, subcutaneously injected B16F10-NDRG2 cells showed delayed tumor growth compared with B16F10-mock cells. Furthermore, severe metastasis from primary tumor mass into the draining lymph nodes was observed after injection of B16F10-mock cells, but not with B16F10-NDRG2 cells. Pulmonary metastasis after intravenous injection of B16F10 cells was also reduced by NDRG2 expression. Intra- and peritumoral angiogenesis that is critical for the tumor growth and metastasis was clearly found in tumors after injection with B16F10-mock cells, whereas it was impaired in tumors after injection with B16F10-NDRG2 cells. Collectively, our data show that NDRG2 expression significantly suppresses tumor invasion by inhibiting MMP activities, which are regulated through the NF-B signaling. Moreover, results from animal experiments provide evidence for the regulatory role of the NDRG2 gene in metastatic tumors.

Nuclear localization of active HGF receptor Met in aggressive MDA-MB231 breast carcinoma cells

Matteucci, E., Bendinelli, P., Desiderio, M. A. — 2009-06-03

Hepatocyte growth factor (HGF)/Met system is deregulated in tumors and is implicated in different aspects of invasive growth. Here, we report that in the highly aggressive MDA-MB231 breast carcinoma cells, Met cytosolic fragments [C-terminal fragment (CTF)] were present in the nuclei. They were constitutively active because tyrosine phosphorylated at regulatory and catalytic domains and endowed with transactivating activity independently of HGF exposure. In fact, various constructs containing juxtamembrane (Jxtm) Met fragments, fused with Gal4 DNA-binding domain, transactivated Gal4Luc activity. MDA-MB231 cells were devoid of WW domain-containing oxidoreductase (Wwox) tumor suppressor. Exogenous Wwox protein expression negatively regulated Jxtm3-transactivating activity and decreased spontaneous migration of MDA-MB231 cells. Also, we demonstrate that the lack of endogenous Wwox in MDA-MB231 cells represented a molecular mechanism for intranuclear Met-CTF accumulation and for the decrease of full-length Met stability. Yes-associated proteins maintained constitutively activated nuclear Met fragments that played a role as transcription factors regulating genes probably including those for motile phenotype. The difference with low invasive MCF-7 cells was evident because the latter did not show intranuclear Met and the transfected constructs-containing Jxtm fragments were inactive also in the presence of HGF. The constitutive activation of nuclear Met-signaling pathway in MDA-MB231 cells, possibly determined at genetic or epigenetic levels of WWOX gene, might participate in breast carcinoma progression influencing invasive/metastatic phenotype. Wwox/Met system can be suggested as a potential target to impair breast carcinoma progression.

Characterization of the metabolic changes underlying growth factor angiogenic activation: identification of new potential therapeutic targets

2009-06-03

Angiogenesis is a fundamental process to normal and abnormal tissue growth and repair, which consists of recruiting endothelial cells toward an angiogenic stimulus. The cells subsequently proliferate and differentiate to form endothelial tubes and capillary-like structures. Little is known about the metabolic adaptation of endothelial cells through such a transformation. We studied the metabolic changes of endothelial cell activation by growth factors using human umbilical vein endothelial cells (HUVECs), [1,2-¹³C₂]-glucose and mass isotopomer distribution analysis. The metabolism of [1,2-¹³C₂]-glucose by HUVEC allows us to trace many of the main glucose metabolic pathways, including glycogen synthesis, the pentose cycle and the glycolytic pathways. So we established that these pathways were crucial to endothelial cell proliferation under vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) stimulation. A specific VEGF receptor-2 inhibitor demonstrated the importance of glycogen metabolism and pentose cycle pathway. Furthermore, we showed that glycogen was depleted in a low glucose medium, but conserved under hypoxic conditions. Finally, we demonstrated that direct inhibition of key enzymes to glycogen metabolism and pentose phosphate pathways reduced HUVEC viability and migration. In this regard, inhibitors of these pathways have been shown to be effective antitumoral agents. To sum up, our data suggest that the inhibition of metabolic pathways offers a novel and powerful therapeutic approach, which simultaneously inhibits tumor cell proliferation and tumor-induced angiogenesis.

The miR-18a* microRNA functions as a potential tumor suppressor by targeting on K-Ras

Tsang, W. P., Kwok, T. T. — 2009-06-03

The Ras proto-oncogene mediates a wide variety of cellular events and is frequently mutated in cancer. MicroRNAs (miRNAs) may regulate the development of cancer through their effect on the target genes. In the search of miRNAs that target on Ras, miR-18a* is the first time confirmed to target on K-Ras and furthermore not on N- and H-Ras. miR-18a* repression by transfection with anti-miR-18a* inhibitor increased the K-Ras expression as well as the luciferase activity of a reporter construct containing the 3'-untranslated region of K-Ras messenger RNA. Furthermore, the miR-18a* repression also increased the cell proliferation and promoted the anchorage-independent growth in soft agar of human squamous carcinoma A431 cells, colon adenocarcinoma HT-29 cells and fetal hepatic WRL-68 cells. On the other hand, ectopic expression of miR-18a* by transfection with miR-18a* precursor suppressed K-Ras expression, cell proliferation and anchorage-independent growth of A431 cells. The increase in cell proliferation and anchorage-independent growth upon miR-18a* repression was, however, rendered by the Ras inhibitor farnesylthiosalicylic acid. In conclusion, miR-18a* may function as a tumor suppressor by targeting on K-Ras. Therefore, the miRNA may also be a potential therapeutic agent or target for cancer therapy.

Novel variants of muscle calpain 3 identified in human melanoma cells: cisplatin-induced changes in vitro and differential expression in melanocytic lesions

Moretti, D., Del Bello, B., Cosci, E., Biagioli, M., Miracco, C., Maellaro, E. — 2009-06-03

Calpains are cysteine proteases comprising members ubiquitously expressed in human tissues and other tissue-specific isoforms. Alterations of calpain 3 (p94), the muscle-specific isoform that contains three peculiar sequences (NS, IS1 and IS2), are strictly associated to the limb-girdle muscular dystrophy type 2A, in which a myonuclear apoptosis has been documented. Our recent demonstration of a proapoptotic role of ubiquitous calpains in drug-induced apoptosis of melanoma cells prompted us to investigate the expression of calpain 3 in human melanoma cell lines undergoing apoptosis and in melanocytic lesions. In melanoma cell lines, we have identified two novel splicing variants of calpain 3 (hMp78 and hMp84): they have an atypical initiation exon and a putative nuclear localization signal, the shorter one lacks IS1 inset and both proteins are extremely unstable. Virtually, both isoforms (prevalently as cleavage forms) are localized in cytoplasm and in nucleoli. In cisplatin-treated preapoptotic cells, an increase of both transcription and autoproteolytic cleavage of the novel variants is observed; the latter event is prevented by the inhibitor of ubiquitous calpains, calpeptin, which is also able to protect from apoptosis. Interestingly, among melanocytic lesions, the expression of these novel variants is significantly downregulated, compared with benign nevi, in the most aggressive ones, i.e. in vertical growth phase melanoma and, even more, in metastatic melanoma cells, characterized by invasiveness properties and usually highly resistant to apoptosis. On the whole, our observations suggest that calpain 3 variants can play a proapoptotic role in melanoma cells and its downregulation, as observed in highly aggressive lesions, could contribute to melanoma progression.

Aromatic DNA adducts and polymorphisms in metabolic genes in healthy adults: findings from the EPIC-Spain cohort

2009-06-03

Aromatic compounds such as polycyclic aromatic hydrocarbons, arylamines and heterocyclic amines require metabolic activation to form metabolites able to bind to DNA, a process mediated by polymorphic enzymes. We measured aromatic DNA adducts in white blood cells by the ³²P-post-labelling assay in a sample of 296 healthy adults (147 men and 149 women) from five regions of Spain. We also analyzed functional polymorphisms in the metabolic genes CYP1A1, CYP1A2, EPHX1, GSTM1, GSTT1, NAT2 and SULT1A1. A significant increased level of DNA aromatic adducts was found related to the fast oxidation–hydrolysis phenotype defined by the polymorphism I462V in CYP1A1, the allele A in IVS1–154C>A of CYP1A2 and the combination Tyrosine–Arginine for Y113H and H139R of EPHX1. Geometric means (adducts per 10^–9 normal nucleotides) were 2.17, 4.04 and 6.30 for slow, normal and fast phenotypes, respectively (P-trend = 0.01). Slow acetylation by NAT2 was associated with a significant decrease in adduct level; subjects with slow alleles *5A and *7A/B had in average 1.56 x 10^–9adducts, as compared with 5.60 for those with normal NAT2 activity (P-value = 0.01). No association was seen with polymorphisms of other metabolic genes such as GSTM1, GSTT1 or SULT1A1. We concluded that the metabolic pathways of oxidation, hydrolysis and acetylation are relevant to the formation of bulky DNA adducts. This could suggest a potential involvement of aromatic compounds in the formation of such adducts; however, given lack of specificity of the post-labeling assay, a firm conclusion cannot be drawn.

Genetic variation in the vitamin C transporter, SLC23A2, modifies the risk of HPV16-associated head and neck cancer

2009-06-03

Human papillomavirus (HPV) type 16 infection is an etiologic factor in a subset of head and neck squamous cell carcinomas (HNSCC). It is unknown if host genetic susceptibility modifies the HPV16–HNSCC association. DNA samples collected as part of a Boston area case–control study of HNSCC were genotyped for single-nucleotide polymorphisms (SNPs) from the National Cancer Institute's SNP500Cancer database. Analysis of demographic, phenotypic and genotypic data for 319 HNSCC cases and 495 frequency-matched controls was performed using unconditional logistic regression. All reported P-values are two sided. We identified a polymorphism in the sodium-dependent vitamin C transporter SLC23A2 that modifies the risk of HNSCC associated with HPV16 infection. Among those with a wild-type allele at SLC23A2, the risk of HNSCC associated with HPV16-positive serology was 5.0 (95% confidence interval (CI) = 3.2–7.8). However, among those with a homozygous variant genotype, the risk of HNSCC associated with HPV16 was attenuated [odds ratio (OR) = 2.8; 95% CI = 1.2–6.2]. Further, when we tested whether genotype modified the interaction between citrus exposure, HPV16, and HNSCC, we found a dramatically increased risk of HNSCC for those with a wild-type SLC23A2 allele, HPV16-positive serology and high citrus intake (OR = 7.4; 95% CI = 3.6–15.1). These results suggest that SLC23A2 genetic variation alters HPV16-associated HNSCC while also highlighting the important role of citrus exposure in this disease.

Association of common genetic variants in SMAD7 and risk of colon cancer

2009-06-03

Two recent genome-wide association studies (GWAS) identified three common variants in SMAD7 (rs4464148, rs4939827 and rs12953717) that confer modest susceptibility to colorectal cancer. Here, we replicated the association of rs4464148 with colon cancer in a population-based case–control study (561 cases and 721 controls). Compared with the TT genotype, those with CT and CC had an adjusted odds ratio (OR) and 95% confidence interval of 1.06 (0.82–1.38) and 1.86 (1.17–2.96), respectively (P_trend = 0.04). However, stratified analyses revealed that this association was limited to women only [OR = 1.25 (0.88–1.78) for CT and OR = 2.76 (1.53–4.98) for CC, P_trend = 0.002, P_interaction = 0.08], which was not noted in any GWAS. Similarly, we found evidence for association with both rs4939827 and rs12953717 in women only (P = 0.007 in dominant rs4939827 model and P = 0.015 in recessive rs12953717 model), but not in men (P > 0.05) and evidence of an interaction with gender (P = 0.015 for rs4939827 and P = 0.061 for rs12953717). Similar effect modification was found in haplotype analyses. Our data add evidence supporting these genetic variants as markers predisposing to colon cancer, specifically in women.

Common genetic variants on 5p15.33 contribute to risk of lung adenocarcinoma in a Chinese population

2009-06-03

Chromosome 5p15.33, containing TERT and CLPTM1L genes, was recently identified as one of the susceptible regions for lung cancer in Caucasian populations. We hypothesized that single-nucleotide polymorphisms (SNPs) identified in this region in Caucasians are also important in the development of lung cancer in Chinese population. To test this hypothesis, we genotyped two most significant SNPs reported in Caucasians, rs2736100A/C and rs402710C/T at 5p15.33, in a case–control study with 1221 non-small cell lung cancer (NSCLC) cases and 1344 cancer-free controls in a Chinese population. We found that rs2736100C allele in TERT gene was associated with a significantly increased risk of NSCLC with adjusted odds ratios of 1.26 [95% confidence interval (CI) = 1.05–1.51] and 1.31 (95% CI = 1.04–1.66) for one or two copies of the variant C allele, respectively. This significant association was more prominent among female (P for heterogeneity: 0.044), non-smokers (P for heterogeneity: 0.054) and/or the subjects with adenocarcinoma (P for heterogeneity: 0.058). However, no significant association was found between rs402710C/T and NSCLC risk. These results suggest that genetic variants in 5p15.33, especially in TERT gene, may also predispose the susceptibility of lung cancer, especially adenocarcinoma, in Chinese population.

Common genetic variants on 8q24 contribute to susceptibility to bladder cancer in a Chinese population

Wang, M., Wang, M., Zhang, W., Yuan, L., Fu, G., Wei, Q., Zhang, Z. — 2009-06-03

A recent genome-wide association study identified two common variants that confer susceptibility to bladder cancer. We hypothesized that these variants are associated with risk of bladder cancer in Chinese populations. We genotyped rs9642880 G>T on 8q24 and rs710521 A>G on 3q28 in a two-stage case–control study of bladder cancer to evaluate the association and further examined the expression of MYC. We found that the rs9642880 G>T, but not the rs710521 A>G polymorphism, was associated with an increased risk of bladder cancer. Compared with the rs9642880 GG genotype, the GT/TT genotypes were associated with an odds ratio of 1.65 (95% confidence interval = 1.25–2.17), and this risk was more pronounced in young men and for low-risk tumors. Additional experiments revealed that the rs9642880 GT/TT genotypes were associated with enhanced levels of both MYC mRNA and protein in bladder tissues. Our findings suggested that the rs9642880 G>T polymorphism on 8q24 was independently associated with the risk of bladder cancer in Chinese populations.

Nucleotide excision repair core gene polymorphisms and risk of second primary malignancy in patients with index squamous cell carcinoma of the head and neck

Zafereo, M. E., Sturgis, E. M., Liu, Z., Wang, L.-E, Wei, Q., Li, G. — 2009-06-03

The nucleotide excision repair (NER) pathway is central in response to damage induced by environmental carcinogens. Efficiency of this pathway, probably genetically determined, may modulate individual risk of developing squamous cell carcinoma of the head and neck (SCCHN) as well as second primary malignancy (SPM) after the index tumor. We hypothesized that common non-synonymous and regulatory single-nucleotide polymorphisms (SNPs) in the NER core genes individually, and more probably collectively, associated with the risk of SPM. We genotyped for seven selected SNPs in 1376 incident SCCHN patients who were prospectively recruited between 1995 and 2006 and followed for SPM development. We found that 110 patients (8%) developed SPM: 43 (39%) second SCCHN; 38 (35%) other tobacco-associated sites and 29 (26%) other non-tobacco-associated sites. The associations of these SNPs with SPM risk were assessed assuming a recessive genetic model. We did not find any significant associations of each or in combination of the seven SNPs with SPM risk in the recessive models. However, when we explored the combined effect based on an alternatively dominant genetic model, we found that the number of observed risk genotypes was associated with a significantly increased SPM risk in a dose-response manner (P = 0.005) and patients with five to seven risk genotypes had a significantly 2.4-fold increased SPM risk compared with patients with zero to two risk genotypes. These findings suggest that a profile of NER core gene polymorphisms might collectively contribute to risk of SPM not in a recessive model but in a dominant model among patients with an index primary SCCHN. These findings need to be validated in future studies with larger sample sizes and longer follow-up time.

A let-7 microRNA-binding site polymorphism in the KRAS 3' UTR is associated with reduced survival in oral cancers

2009-06-03

MicroRNA (miRNA)-binding site polymorphisms that could contribute to disease risk and prognosis are rapidly being identified and investigated as this genetic variation may have a potentially profound impact on human health. A recently described variant allele in the KRAS 3' untranslated region that arises in the let-7 miRNA complementary site (KRAS-LCS6) and leads to increased KRAS expression in lung cancer was examined for its association with the occurrence of head and neck squamous cell carcinoma (HNSCC). We examined the prevalence of the KRAS-LCS6 variant allele in a population-based case–control study of HNSCC to determine if this KRAS-LCS6 genotype was associated with disease occurrence and patient survival. Although the KRAS-LCS6 variant genotype was not associated with the overall risk of HNSCC, cases with the KRAS-LCS6 variant genotype had significantly reduced survival [hazard ratio (HR), 1.6; 95% confidence interval (CI), 1.0–2.5] in models controlled for confounders of survival. This risk was greatest in cases of oral cavity carcinoma (HR, 2.7; 95% CI, 1.4–5.3). These data demonstrate that cases with the KRAS-LCS6 variant have significantly reduced survival time and suggest that this variant may alter the phenotype or therapeutic response of this disease.

Differential effects of several phytochemicals and their derivatives on murine keratinocytes in vitro and in vivo: implications for skin cancer prevention

2009-06-03

The purpose of our study was to investigate in vitro the potential cancer preventive properties of several phytochemicals, i.e. grape seed extract (GSE), resveratrol (RES), ursolic acid (URA), ellagic acid (ELA), lycopene and N-acetyl-L-cysteine (NAC) to define the mechanisms by which these compounds may inhibit murine skin carcinogenesis. We measured quenching of peroxyl, superoxide and hydroxyl radicals by these phytochemicals. We also used adenosine triphosphate (ATP) bioluminescence, Caspase-Glo 3/7 and P450-Glo (CYP1A1 and CYP1B1) assays to study antiproliferative, proapoptotic and CYP-inhibiting effects of the phytochemicals. We next determined their effects on a 4 week inflammatory hyperplasia assay using 7,12-dimethylbenz[a]anthracene-induced murine skin carcinogenesis model to further understand their mechanism of action. Three murine keratinocyte cell lines, i.e. non-tumorigenic (3PC), papilloma-derived (MT1/2) and squamous cell carcinoma-derived (Ca3/7) cell lines, were used in in vitro assays. We have found that GSE, ELA and RES are potent scavengers of peroxyl and superoxide radicals. Statistically significant effects on activities of caspase-3 and -7 were observed only after GSE and URA treatments. All tested compounds protected cells from hydrogen peroxide-induced DNA damage. Using a short-term complete carcinogenesis assay, we have found that all selected compounds caused marked decreases of epidermal thickness and (except RES) reduced percentages of mice with mutation in codon 61 of Ha-ras oncogene. In conclusion, differential effects of tested phytochemicals on events and processes critical for the growth inhibition of keratinocytes in vitro and in vivo indicate that combinations of tested compounds may, in the future, better counteract both tumor initiation and tumor promotion/progression.

Lung tumor promotion by curcumin

2009-06-03

Curcumin exhibits anti-inflammatory and antitumor activity and is being tested in clinical trials as a chemopreventive agent for colon cancer. Curcumin's chemopreventive activity was tested in a transgenic mouse model of lung cancer that expresses the human Ki-ras^G12C allele in a doxycycline (DOX) inducible and lung-specific manner. The effects of curcumin were compared with the lung tumor promoter, butylated hydroxytoluene (BHT), and the lung cancer chemopreventive agent, sulindac. Treatment of DOX-induced mice with dietary curcumin increased tumor multiplicity (36.3 ± 0.9 versus 24.3 ± 0.2) and progression to later stage lesions, results which were similar to animals that were co-treated with DOX/BHT. Microscopic examination showed that the percentage of lung lesions that were adenomas and adenocarcinomas increased to 66% in DOX/BHT, 66% in DOX/curcumin and 49% in DOX/BHT/curcumin-treated groups relative to DOX only treated mice (19%). Immunohistochemical analysis also showed increased evidence of inflammation in DOX/BHT, DOX/curcumin and DOX/BHT/curcumin mice relative to DOX only treated mice. In contrast, co-treatment of DOX/BHT mice with 80 p.p.m. of sulindac inhibited the progression of lung lesions and reduced the inflammation. Lung tissue from DOX/curcumin-treated mice demonstrated a significant increase (33%; P = 0.01) in oxidative damage, as assessed by the levels of carbonyl protein formation, relative to DOX-treated control mice after 1 week on the curcumin diet. These results suggest that curcumin may exhibit organ-specific effects to enhance reactive oxygen species formation in the damaged lung epithelium of smokers and ex-smokers. Ongoing clinical trials thus may need to exclude smokers and ex-smokers in chemopreventive trials of curcumin.

Genetic versus chemoprotective activation of Nrf2 signaling: overlapping yet distinct gene expression profiles between Keap1 knockout and triterpenoid-treated mice

2009-06-03

Loss of NF-E2-related factor 2 (Nrf2) signaling increases susceptibility to acute toxicity, inflammation and carcinogenesis in mice due to the inability to mount adaptive responses. In contrast, disruption of Keap1 (a cytoplasmic modifier of Nrf2 turnover) protects against these stresses in mice, although inactivating mutations in Keap1 have been identified recently in some human cancers. Global characterization of Nrf2 activation is important to exploit this pathway for chemoprevention in healthy, yet at-risk individuals and also to elucidate the consequences of hijacking the pathway in Keap1-mutant human cancers. Liver-targeted conditional Keap1-null, Albumin-Cre:Keap1^(flox/–) (CKO) mice provide a model of genetic activation of Nrf2 signaling. By coupling global gene expression analysis of CKO mice with analysis of pharmacologic activation using the synthetic oleanane triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), we are able to gain insight into pathways affected by Nrf2 activation. CDDO-Im is an extremely potent activator of Nrf2 signaling. CKO mice were used to identify genes modulated by genetic activation of Nrf2 signaling. The CKO response was compared with hepatic global gene expression changes in wild-type mice treated with CDDO-Im at a maximal Nrf2 activating dose. The results show that genetic and pharmacologic activation of Nrf2 signaling modulates pathways beyond detoxication and cytoprotection, with the largest cluster of genes associated with lipid metabolism. Genetic activation of Nrf2 results in much larger numbers of detoxication and lipid metabolism gene changes. Additionally, analysis of pharmacologic activation suggests that Nrf2 is the primary mediator of CDDO-Im activity, though other cell-signaling targets are also modulated following an oral dose of 30 µmol/kg.

Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint

2009-06-03

Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2–160 µg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs spindle checkpoint signaling, which may contribute to the antiproliferative effects of the compound.

Promoter CpG island hypermethylation- and H3K9me3 and H3K27me3-mediated epigenetic silencing targets the deleted in colon cancer (DCC) gene in colorectal carcinogenesis without affecting neighboring genes on chromosomal region 18q21

2009-06-03

Chromosomal loss of 18q21 is a frequent event in colorectal cancer (CRC) development, suggesting that this region harbors tumor suppressor genes (TSGs). Several candidate TSGs, among which methyl-CpG-binding domain protein 1 (MBD1), CpG-binding protein CXXC1, Sma- and Mad-related protein 4 (SMAD4), deleted in colon cancer (DCC) and methyl-CpG-binding domain protein 2 (MBD2) are closely linked on a 4-Mb DNA region on chromosome18q21. As TSGs can be epigenetically silenced, this study investigates whether MBD1, CXXC1, SMAD4, DCC and MBD2 are subject to epigenetic silencing in CRC. Methylation-specific polymerase chain reaction and sodium bisulfite sequencing of these genes show that DCC, but not MBD1, CXXC1, SMAD4 and MBD2, has promoter CpG island methylation in CRC cell lines and tissues {normal mucosa [29.5% (18/61)], adenomas [81.0% (47/58)] and carcinomas [82.7% (62/75)] (P = 8.6 x 10^–9)} that is associated with reduced DCC expression, independent of 18q21 loss analyzed by multiplex ligation-dependent probe amplification. Reduced gene expression of CXXC1, SMAD4 and MBD2 correlates with 18q21 loss in CRC cell lines (P = 0.04, 0.02 and 0.02, respectively). Treatment with the demethylating agent 5-aza-2'-deoxycytidine, but not with the histone deacetylase inhibitor trichostatin A exclusively restored DCC expression in CRC cell lines. Chromatin immunoprecipitation studies reveal that the DCC promoter is marked with repressive histone-tail marks H3K9me3 and H3K27me3, whereas activity related H3K4me3 was absent. Only active epigenetic marks were detected for MBD1, CXXC1, SMAD4 and MBD2. This study demonstrates specific epigenetic silencing of DCC in CRC as a focal process not affecting neighboring genes on chromosomal region 18q21.

Interaction between HSP60 and {beta}-catenin promotes metastasis

2009-06-03

Heat shock protein 60 (HSP60) plays an essential role in assisting many newly synthesized proteins to reach their native forms. Increased HSP60 expression is observed in different types of human cancers with metastasis (e.g. pancreatic cancer and large bowel carcinoma). However, the role of HSP60 in metastasis remains little known. Aberrant activation of β-catenin plays a key role in tumorigenesis and metastasis. Here, we show that overexpression of HSP60 induces metastatic phenotypes in vitro and in vivo. HSP60 interacts with β-catenin, increases β-catenin protein levels through the apical domain and enhances its transcriptional activity. Short-interference RNA-mediated repression of β-catenin reverts metastatic activity caused by HSP60 overexpression. Proteosomal activity is not required for the induction of β-catenin by HSP60. Coexpression of HSP60 and nuclear β-catenin predicts a worse prognosis of metastatic head and neck cancer patients. These results implicate a novel role of HSP60 in metastasis.

Endocrine dysfunction in p27Kip1 deficient mice and susceptibility to Wnt-1 driven breast cancer

Glover, C. E., Gurley, K. E., Kim, K.-H., Storer, B., Fero, M. L., Kemp, C. J. — 2009-06-03

The cyclin-dependent kinase (Cdk) inhibitor p27^Kip1 (p27) is a marker of prognosis in many cancers, including breast cancer. Low p27 expression correlates with poor prognosis, especially in hormone receptor positive breast tumors. This association suggests a role for p27 in hormone-dependent cancer. We used the Wnt-1 transgenic mouse model to further explore the role of p27 in hormone-driven breast cancer. We found that p27 deficiency did not alter breast cancer rate in either male or female Wnt-1 mice. However, we did find p27–/– females had reduced levels of serum progesterone (P) and increased variability in estradiol (E), which could have affected their cancer susceptibility. To equalize hormone levels, an additional cohort of Wnt-1 female mice was ovariectomized and implanted with slow release pellets of E and P. Although this treatment did not alter the breast cancer rate, it did accelerate the development of pituitary and gastric tumors in p27–/– mice. This study shows that while not a significant inhibitor of Wnt-1-driven breast cancer, p27 inhibits gastric tumors, whose latency is modulated by sex steroids.

Differential effects of arsenic on cutaneous and systemic immunity: focusing on CD4+ cell apoptosis in patients with arsenic-induced Bowen's disease

2009-06-03

Bowen's disease (BD), a carcinoma in situ of the skin, has been identified as an early lesion in arsenic carcinogenesis. Patients with arsenic-induced Bowen's disease (As-BD) showed both cutaneous and systemic immune dysfunctions. We set out to evaluate the interactions between keratinocytes and lymphocytes in the context of As-BD carcinogenesis. Our results showed that As-BD lesions demonstrated a significant dermal CD4+ cell, an essential regulator of proper tumor immunity, undergoing apoptosis. In addition, it was found that the As-BD patients have lower percentage of peripheral CD4+ cells as compared with control subjects. However, the CD4+ cells from As-BD patients were less susceptible to arsenic-induced apoptosis, due to reduced tumor necrosis factor receptor 1 expression. Interestingly, arsenic was found to induce Fas expression on CD4+ cells and increase the soluble Fas ligand (sFasL) production from keratinocytes. This sFasL-containing keratinocyte supernatant was able to induce comparable CD4+ cell apoptosis for both patients and controls. Using immunofluorescent staining, increased FasL was observed in keratinocytes of As-BD lesions and Fas was expressed among infiltrating CD4+ cells. Our findings suggested that systemically, the percentage of CD4+ cells was decreased in the peripheral blood of As-BD patients. These residual CD4+ cells were less susceptible to arsenic-induced apoptosis. However, once infiltrated into the As-BD lesions, the selective CD4+ cell apoptosis might be mediated by FasL from keratinocytes. This additional tumor-anti-immune phenomenon present in the cutaneous environment provides a reasonable explanation for frequent occurrence of arsenic cancers in the skin.

Frontmatter

2009-04-30

Backmatter

2009-04-30

Super competition as a possible mechanism to pioneer precancerous fields

Rhiner, C., Moreno, E. — 2009-04-30

Cancer is the result of sequential genetic changes over time that transform a cell into a malignant and ultimately invasive entity. The insight that cancerous cells arise from a series of mutations in oncogenes and tumor suppressors, commonly known as multistep carcinogenesis, has been conceptually elaborated and proven in the last 20 years. Although knowledge about late steps of cancerogenesis and disease progression has greatly advanced, the initial molecular events remain largely unknown. Basic research in Drosophila has started the quest to find early markers that detect initial clonal expansion of precancerous cells. These efforts were spurred by novel findings demonstrating that certain mutations transform cells into super-competitors that expand at the expense of the surrounding epithelial cells without inducing histological changes. This mechanism, discovered as super competition in the fly, might also lie at the heart of a clinical observation termed ‘field cancerization’. This review aims to bring together current understanding from basic research on cell competition and clinical studies that have analyzed field characteristics to highlight parallels and possible connections.

Rottlerin induces apoptosis via death receptor 5 (DR5) upregulation through CHOP-dependent and PKC {delta}-independent mechanism in human malignant tumor cells

Lim, J. H., Park, J.-W., Choi, K. S., Park, Y. B., Kwon, T. K. — 2009-04-30

Rottlerin has been shown to induce antiproliferation and apoptosis of human cancer cell lines. In this study, we demonstrate a novel mechanism of rottlerin-induced apoptosis via death receptor (DR) 5 upregulation. We found that treatment with rottlerin significantly induces DR5 expression both at its messenger RNA and protein levels. Downregulation of DR5 expression with small-interfering RNA (siRNA) efficiently attenuated rottlerin-induced apoptosis, showing that the critical role of DR5 in this cell death. Rottlerin-induced DR5 upregulation was accompanied by CCAAT/enhancer-binding protein–homologous protein (CHOP) protein expression and rottlerin-induced increase of DR5 promoter activity was diminished by mutation of a CHOP-binding site of DR5 promoter. Although rottlerin is known to be as an inhibitor of novel isoforms of protein kinase C (PKC), specifically PKC , not only suppression of PKC expression by siRNA but also overexpression of wild-type-PKC or dominant-negative-PKC did not affect the rottlerin-mediated induction of DR5 in our study. These results suggest that rottlerin induces upregulation of DR5 via PKC -independent pathway. Furthermore, subtoxic dose of rottlerin sensitizes human cancer cells, but not normal cells, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Thus, DR5-mediated apoptosis, which is induced by rottlerin alone or by the combined treatment with rottlerin and TRAIL, may offer a new therapeutic strategy against cancer.

ATP-sensitive potassium channels control glioma cells proliferation by regulating ERK activity

Huang, L., Li, B., Li, W., Guo, H., Zou, F. — 2009-04-30

Ion channels are found in a variety of cancer cells and necessary for cell cycle and cell proliferation. The roles of K⁺ channels in the process are, however, poorly understood. In the present study, we report that adenosine triphosphate (ATP)-sensitive potassium channel activity plays a critical role in the proliferation of glioma cells. The expression of K_ATP channels in glioma tissues was greatly increased than that in normal tissues. Treatment of glioma cells with tolbutamide, K_ATP channels inhibitor, suppressed the proliferation of glioma cells and blocked glioma cell cycle in G₀/G₁ phase. Similarly, downregulation of K_ATP channels by small interfering RNA (siRNA) inhibited glioma cell proliferation. On the other hand, K_ATP channels agonist diazoxide and overexpression of K_ATP channels promoted the proliferation of glioma cells. Moreover, inhibiting K_ATP channels slowed the formation of tumor in nude mice generated by injection of glioma cells. Whereas activating K_ATP channels promoted development of tumor in vivo. The effect of K_ATP channels activity on glioma cells proliferation is mediated by extracellular signal-regulated kinase (ERK) activation. We found that activating K_ATP channel triggered ERK activation and inhibiting K_ATP channel depressed ERK activation. U-0126, the mitogen-activated protein kinase kinase (MAPK kinase) inhibitors blocked ERK activation and cell proliferation induced by diazoxide. Furthermore, constitutively activated MEK plasmids transfection reversed the inhibitory effects of tolbutamide on glioma proliferation, lending further support for a role of ERK in mediating this process. Our results suggest that K_ATP channels control glioma cell proliferation via regulating ERK pathway. We concluded that K_ATP channels are important in pathological cell proliferation and open a promising pathway for novel targeted therapies.

Ceramide synthases and ceramide levels are increased in breast cancer tissue

2009-04-30

Several in vitro studies have correlated dysfunction of the sphingolipid-signaling pathway with promotion of tumor cell growth as well as progression and resistance of tumors to chemotherapeutic agents. As ceramides (Cer) constitute the structural backbones of all sphingolipids, we investigated the endogenous ceramide levels in 43 malignant breast tumors and 21 benign breast biopsies and compared them with those of normal tissues using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The total ceramide levels in malignant tumor tissue samples were statistically significantly elevated when compared with normal tissue samples. Upregulation of the total ceramide level averaged 12-fold and 4-fold higher than normal tissue samples, for malignant tumors and benign tissues, respectively. Specifically, the levels of C_16:0-Cer, C_24:1-Cer and C_24:0-Cer were significantly raised in malignant tumors as compared with benign and normal tissue. The augmentation of the various ceramides could be assigned to an increase of the messenger RNA levels of ceramide synthases (CerS) LASS2 (longevity assurance), LASS4 and LASS6. Notably, elevated levels of C_16:0-Cer were associated with a positive lymph node status, indicating a metastatic potential for this ceramide. Moreover, the levels of C_18:0-Cer and C_20:0-Cer were significantly higher in estrogen receptor (ER) positive tumor tissues as compared with ER negative tumor tissues. In conclusion, progression in breast cancer is associated with increased ceramide levels due to an upregulation of specific LASS genes.

Involvement of NF-{kappa}B and AP-1 in COX-2 upregulation by human papillomavirus 16 E5 oncoprotein

Kim, S.-H., Oh, J.-M., No, J.-H., Bang, Y.-J., Juhnn, Y.-S., Song, Y.-S. — 2009-04-30

The human papillomavirus (HPV) E6 and E7 oncoproteins play important roles in cervical carcinogenesis through multiple mechanisms, including upregulation of cyclooxygenase-2 (COX-2), which has been shown to be involved in both carcinogenesis and cancer progression. To explore the role of E5 in cervical carcinogenesis, we herein investigated the effect of HPV 16 E5 on COX-2 expression. Our results revealed that E5 induced COX-2 expression through the epidermal growth factor receptor-signaling pathway, with nuclear factor-kappaB (NF-B) and activator protein-1 (AP-1) acting as critical factors in E5-induced COX-2 expression. NF-B inhibition blocked COX-2 expression more potently than inhibition of AP-1. Our findings collectively suggest that the HPV 16 E5 oncoprotein mediates cervical carcinogenesis at least in part via upregulation of COX-2 expression through NF-B and AP-1, with NF-B playing a larger role.

Genetic polymorphisms in the cytokine genes and risk of hepatocellular carcinoma in low-risk non-Asians of USA

Ognjanovic, S., Yuan, J.-M., Chaptman, A. K., Fan, Y., Yu, M. C. — 2009-04-30

Polymorphisms in cytokine genes responsible for inflammatory and immune responses are associated with risk of hepatocellular carcinoma (HCC) in high-risk Chinese population. Similar data in low-risk populations are lacking. A population-based case–control study of HCC was conducted including 120 HCC patients and 230 matched control subjects of non-Asian residents in Los Angeles County, California. Genetic variants in the interferon (IFN), tumor necrosis factor- (TNF), interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-12 and IL-18 genes were determined by Taqman assays. The logistic regression method was used to analyze the data. For T helper (Th) 1 genes (IFN, IL-6 and IL-12), relative to the putative high-activity genotypes, individual low-activity genotypes were associated with statistically non-significant increases in HCC risk. The odds ratio (OR) was 1.53 [95% confidence interval (CI) = 0.53–4.39] for three versus zero low-activity genotypes. For Th2 cytokines (IL-4 and IL-10), low- versus high-activity genotypes were associated with statistically non-significant decreases in HCC risk. The OR was 0.64 (95% CI = 0.27–1.55) for two versus zero low-activity genotypes. When the Th1 and Th2 genotypes were examined simultaneously, the highest level of risk was observed in individuals jointly possessing the highest number of low-activity Th1 genotypes and the lowest number of low-activity Th2 genotypes. There was a roughly doubling of risk between these two extreme genetic profiles, which did not reach statistical significance (OR = 1.98, 95% CI = 0.50–7.84, P = 0.08). In contrast to high-risk Chinese, Th1 and Th2 genotypes did not impact in a major way on risk of HCC in USA non-Asians.

Genetic polymorphisms in 85 DNA repair genes and bladder cancer risk

2009-04-30

Several defense mechanisms have been developed and maintained during the evolution to protect human cells against damage produced from exogenous or endogenous sources. We examined the associations between bladder cancer and a panel of 652 polymorphisms from 85 genes involved in maintenance of genetic stability [base excision repair, nucleotide excision repair, double-strand break repair (DSBR) and mismatch repair, as well as DNA synthesis and cell cycle regulation pathways] in 201 incident bladder cancer cases and 326 hospital controls. Score statistics were used to test differences in haplotype frequencies between cases and controls in an unconditional logistic regression model. To account for multiple testing, we associated to each P-value the expected proportion of false discoveries (q-value). Haplotype analysis revealed significant associations (P < 0.01) between bladder cancer and two genes (POLB and FANCA) with an associated q-value of 24%. A permutation test was also used to determine whether, in each pathway analyzed, there are more variants whose allelic frequencies are different between cases and controls as compared with what would be expected by chance. Differences were found for cell cycle regulation (P = 0.02) and to a lesser extent for DSBR (P = 0.05) pathways. These results hint to a few potential candidate genes; however, our study was limited by the small sample size and therefore low statistical power to detect associations. It is anticipated that genome-wide association studies will open new perspectives for interpretation of the results of extensive candidate gene studies such as ours.

Vitamin D-related genes, serum vitamin D concentrations and prostate cancer risk

2009-04-30

We systematically investigated the association of 48 SNPS in four vitamin D metabolizing genes [CYP27A1, GC, CYP27B1 and CYP24A1] with serum 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)₂D] levels and the association of these SNPS and an additional 164 SNPS in eight downstream mediators of vitamin D signaling [VDR, RXRA, RXRB, PPAR, NCOA1, NCOA2, NCOA3 and SMAD3] with prostate cancer risk in the 749 incident prostate cancer cases and 781 controls of the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. 25(OH)D (all cases and controls) and 1,25(OH)₂D (a subset of 150 controls) levels were measured by radioimmunoassay and SNP data were genotyped as part of a genome-wide scan. Among investigated SNPS, only four tag SNPS in GC, the major serum 25(OH)D carrier, were associated with 25(OH)D levels; no SNPS were associated with 1,25(OH)₂D levels. None of the 212 SNPS examined were associated with cancer risk overall. Among men in the lowest tertile of serum 25(OH)D (<48.9 nmol/l), however, prostate cancer risk was related to tag SNPS in or near the 3' untranslated region (UTR) of VDR, with the strongest association for rs11574143 [odds ratio (95% confidence interval) for risk allele carriers versus wild-type: 2.49 (1.51–4.11), P = 0.0007]; the genotype associations were null among men in tertile 2 and tertile 3. Results from the most comprehensive evaluation of serum vitamin D and its related genes to date suggest that tag SNPS in the 3' UTR of VDR may be associated with risk of prostate cancer in men with low vitamin D status.

Oxidative stress-related genotypes, fruit and vegetable consumption and breast cancer risk

2009-04-30

Dietary antioxidants may interact with endogenous sources of pro- and antioxidants to impact breast cancer risk. A nested case–control study of postmenopausal women (505 cases and 502 controls) from the Cancer Prevention Study-II Nutrition Cohort was conducted to examine the interaction between oxidative stress-related genes and level of vegetable and fruit intake on breast cancer risk. Genetic variations in catalase (CAT) (C–262T), myeloperoxidase (MPO) (G–463A), endothelial nitric oxide synthase (NOS3) (G894T) and heme oxygenase-1 (HO-1) [(GT)_n dinucleotide length polymorphism] were not associated with breast cancer risk. Women carrying the low-risk CAT CC [odds ratio (OR) = 0.75, 95% confidence interval (CI) 0.50–1.11], NOS3 TT (OR = 0.54, 95% CI = 0.26–1.12, P-trend = 0.10) or HO-1 S allele and MM genotype (OR = 0.56, 95% CI = 0.37–0.55), however, were found to be at non-significantly reduced breast cancer risk among those with high vegetable and fruit intake (≥median; P-interactions = 0.04 for CAT, P = 0.005 for NOS3 and P = 0.07 for HO-1). Furthermore, those with ≥4 putative low-risk alleles in total had significantly reduced risk (OR = 0.53, 95% CI = 0.32–0.88, P-interaction = 0.006) compared with those with ≤2 low-risk alleles. In contrast, among women with low vegetable and fruit intake (< median), the low-risk CAT CC (OR = 1.33, 95% CI = 0.89–1.99), NOS3 TT (OR = 2.93, 95% CI = 1.38–6.22) and MPO AA (OR = 2.09, 95% CI = 0.73–5.95) genotypes appeared to be associated with raised breast cancer risk, with significantly increased risks observed in those with ≥4 low-risk alleles compared with participants with ≤2 low-risk alleles (OR = 1.77, 95% CI = 1.05–2.99, P-interaction = 0.006). Our results support the hypothesis that there are joint effects of endogenous and exogenous antioxidants.

Genetic susceptibility to esophageal cancer: the role of the nucleotide excision repair pathway

2009-04-30

In this case–control study with 387 White esophageal patients and 462 White controls matched to cases by age and sex, we evaluated the associations between 13 potential functional polymorphisms in eight major nucleotide excision repair (NER) genes and esophageal cancer risk. In individual single nucleotide polymorphism analysis, after adjustment for multiple comparisons, the heterozygous GT genotype of the ERCC1 3' untranslated region (UTR) was associated with an increased risk, whereas the homozygous variant genotype TT was associated with 60% reduction in risk with an odds ratio (OR) of 0.40 (95% confidence interval [CI] = 0.19–0.86). The heterozygous AG genotype of XPA 5' UTR was at 2.11-fold increased risk (95% CI = 1.33–3.35) and the risk reached 3.10-fold (95% CI = 1.94–4.95) for the homozygous variant GG genotype. These associations were also significant when restricted the analyses in patients with esophageal adenocarcinoma. Further, the CT genotype of the RAD23B Ala249Val was associated with increased esophageal cancer risk (OR = 1.44; 95% CI = 1.05–1.97), whereas the poly-AT–/+ genotype of the XPC intron 9 conferred a decreased risk (OR = 0.71, 95% CI = 0.51–0.97). In joint analysis, individuals carrying 1 (OR = 2.64, 95% CI = 1.57–4.52) and ≥2 (OR = 2.74, 95% CI = 1.58–4.75) unfavorable genotypes exhibited significantly increased risk for esophageal cancer risk with significant dose-response trend (P for trend = 0.006). The pathway-based risk was more evident in ever smokers, overweight/obese individuals, men and ever drinkers. Our results support the hypothesis that increasing numbers of unfavorable genotypes in the NER predispose susceptible individuals to increased risk of esophageal cancer. These findings warrant further replications in different populations.

Matrix metalloproteinase 1, 3 and 12 polymorphisms and esophageal adenocarcinoma risk and prognosis

2009-04-30

The matrix metalloproteinase (MMP) family degrade extracellular matrix and mediate pathways including apoptosis, angiogenesis and immunity. We studied the association between four MMP polymorphisms within three MMP genes and esophageal adenocarcinoma (EA) risk and prognosis. A total of 313 EA cases and 455 age and gender frequency-matched controls were genotyped for MMP1 1G/2G, MMP3 6A/5A, MMP12 –82A/G and MMP12 1082A/G. The association between individual MMP polymorphisms and EA risk was evaluated using regression models and adjusted for age, gender, adult body mass index and smoking status. Haplotype analysis was performed to investigate the combined effect of all four linked MMP polymorphisms and EA risk. The MMP1 and MMP3 polymorphisms were associated with increased EA risk: MMP1 1G/2G and 2G/2G had adjusted odds ratios of 1.46 [95% confidence interval 1.0–2.1; P = 0.04] and adjusted odds ratio 1.83 (1.2–2.8; P = 0.005), respectively, whereas MMP3 6A/5A had adjusted odds ratio 1.40 (95% confidence interval 1.0–2.1; P = 0.09) and MMP3 5A/5A had 1.61 (95% confidence interval 1.0–2.5; P = 0.03). Two MMP haplotypes [MMP1–MMP3–MMP12 (–82) 2G-5A-A (adjusted odds ratio 1.36, 95% confidence interval 1.0–1.8; P = 0.03) and 2G-5A-G (adjusted odds ratio 1.70, 95% confidence interval 1.1–2.6; P = 0.01)] were also associated with increased EA risk. The relationship between BE cases with the same set of controls was similar. No association was identified between the MMP polymorphisms and overall survival or progression free survival of patients with EA. MMP1, MMP3 and possibly MMP12 –82A/G polymorphisms and their haplotypes are associated with increased EA risk.

Aldose reductase deficiency in mice prevents azoxymethane-induced colonic preneoplastic aberrant crypt foci formation

Tammali, R., Reddy, A. B. M., Ramana, K. V., Petrash, J. M., Srivastava, S. K. — 2009-04-30

Aldose reductase (AR; EC 1.1.1.21), an nicotinamide adenine dinucleotide phosphate-dependent aldo–keto reductase, has been shown to be involved in oxidative stress signaling initiated by inflammatory cytokines, chemokines and growth factors. Recently, we have shown that inhibition of this enzyme prevents the growth of colon cancer cells in vitro as well as in nude mice xenografts. Herein, we investigated the mediation of AR in the formation of colonic preneoplastic aberrant crypt foci (ACF) using azoxymethane (AOM)-induced colon cancer mice model. Male BALB/c mice were administrated with AOM without or with AR inhibitor, sorbinil and at the end of the protocol, all the mice were euthanized and colons were evaluated for ACF formation. Administration of sorbinil significantly lowered the number of AOM-induced ACF. Similarly, AR-null mice administered with AOM demonstrated significant resistance to ACF formation. Furthermore, inhibition of AR or knockout of AR gene in the mice significantly prevented AOM-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins as well as their messenger RNA. AR inhibition or knockdown also significantly decreased the phosphorylation of protein kinase C (PKC) β2 and nuclear factor kappa binding protein as well as expression of preneoplastic marker proteins such as cyclin D1 and β-catenin in mice colons. Our results suggest that AR mediates the formation of ACF in AOM-treated mice and thereby inhibition of AR could provide an effective chemopreventive approach for the treatment of colon cancer.

Lupeol inhibits proliferation of human prostate cancer cells by targeting {beta}-catenin signaling

2009-04-30

Lupeol, a dietary triterpene, was shown to decrease serum prostate-specific antigen levels and inhibit the tumorigenicity of prostate cancer (CaP) cells in vivo. Here, we show that Lupeol inhibits the proliferative potential of CaP cells and delineated its mechanism of action. Employing a focused microarray of human CaP-associated genes, we found that Lupeol significantly modulates the expression level of genes such as ERBB2, tissue inhibitor of metalloproteinases-3, cyclin D1 and matrix metalloproteinase (MMP)-2 that are known to be associated with proliferation and survival. A common feature of these genes is that all of them are known to either regulate or act as downstream target of β-catenin signaling that is highly aberrant in CaP patients. Lupeol treatment significantly (1) reduced levels of β-catenin in the cytoplasmic and nuclear fractions, (2) modulated expression levels of glycogen synthase kinase 3 beta (GSK3β)–axin complex (regulator of β-catenin stability), (3) decreased the expression level and enzymatic activity of MMP-2 (downstream target of β-catenin), (4) reduced the transcriptional activation of T Cell Factor (TCF) responsive element (marker for β-catenin signaling) in pTK-TCF-Luc-transfected cells and (5) decreased the transcriptional activation of MMP-2 gene in pGL2-MMP-2-Luc-transfected cells. Effects of Lupeol treatment on β-catenin degradation were significantly reduced in CaP cells where axin is knocked down through small interfering RNA transfection and GSK3β activity is blocked. Collectively, these data suggest the multitarget efficacy of Lupeol on β-catenin-signaling network thus resulting in the inhibition CaP cell proliferation. We suggest that Lupeol could be developed as an agent for chemoprevention as well as chemotherapy of human CaP.

Penta-O-galloyl-beta-D-glucose induces S- and G1-cell cycle arrests in prostate cancer cells targeting DNA replication and cyclin D1

Hu, H., Zhang, J., Lee, H. J., Kim, S.-H., Lu, J. — 2009-04-30

We have recently shown that penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG), a naturally occurring hydrolyzable gallotannin, inhibited the in vivo growth of human androgen-independent p53-mutant DU145 prostate cancer (PCa) xenograft in athymic nude mice without adverse effect on their body weight. We have also shown that PGG induced caspase-mediated apoptosis in the DU145 cells and the androgen-dependent human p53-wild-type LNCaP cells. Here, we investigated the cell cycle effects of PGG in these and other PCa cells. Our data show that treatment with subapoptotic doses of PGG induced S-arrest, whereas higher doses of PGG induced not only S-arrest but also G₁ arrest. We show, for the first time, that irrespective of the p53 functional status of the PCa cell lines, PGG exerted a rapid (within 2 h) and potent inhibition (inhibitory concentration by 50% ~6 µM) of 5-bromo-2'-deoxyuridine incorporation into S phase cells. In isolated nuclei, PGG inhibited DNA replicative synthesis with superior efficacy than a known DNA polymerase alpha inhibitor, aphidocolin. In addition to the S-arrest action, we have found a close association of downregulation of cyclin D1 with G₁ arrest induced by PGG. Overexpressing this G₁ cyclin abolished G₁ arrest, but hastened the S-arrest induction by PGG. Together, our data indicate that PGG induced PCa S-arrest probably through DNA replicative blockage and induced G₁ arrest via cyclin D1 downregulation to contribute to anticancer activity. Our data raise the hypothesis that PGG may be a novel inhibitor of DNA polymerases.

PLAB induction in fenretinide-induced apoptosis of ovarian cancer cells occurs via a ROS-dependent mechanism involving ER stress and JNK activation

Appierto, V., Tiberio, P., Villani, M. G., Cavadini, E., Formelli, F. — 2009-04-30

Fenretinide [N-(4-hydroxyphenyl)-retinamide (4HPR)] is a synthetic retinoid with antitumor activity that induces apoptosis in various types of cancer cell. We showed previously that 4HPR upregulates the proapoptotic gene placental bone morphogenetic protein (PLAB), which is a mediator of 4HPR-induced apoptosis in ovarian cancer cells. Here, we investigated the signaling cascade involving PLAB that mediates the apoptotic effect. In 4HPR-sensitive ovarian cancer cells, 4HPR-induced reactive oxygen species (ROS) are involved in PLAB upregulation and apoptosis, both events abrogated by the antioxidants vitamin C and butylated hydroxyanisole. We analyzed the expression and activation of endoplasmic reticulum (ER) stress-associated molecules and show that 4HPR-induced ER stress is a consequence of ROS generation. Salubrinal, an ER stress inhibitor, abrogated 4HPR-induced PLAB upregulation and protected the cells from apoptosis. Downstream of ROS generation and ER stress, 4HPR activated c-Jun N-terminal kinase (JNK), which was inhibited by vitamin C and salubrinal. The JNK inhibitor SP600125 reduced 4HPR-induced PLAB upregulation, by decreasing PLAB mRNA half-life, and protected the cells from apoptosis. These data indicate that 4HPR-induced PLAB upregulation occurs downstream of a signaling cascade involving ROS generation, ER stress induction and JNK activation and that these steps are mediators of 4HPR-induced apoptosis.

C/EBP{beta} regulates body composition, energy balance-related hormones and tumor growth

2009-04-30

The prevalence of obesity, an established epidemiologic risk factor for many chronic diseases including cancer, has been steadily increasing in the US over several decades. The mechanisms used to regulate energy balance and adiposity and the relationship of these factors to cancer are not completely understood. Here we have used knockout mice to examine the roles of the transcription factors CCAAT/enhancer-binding protein (C/EBP) β and C/EBP in regulating body composition and systemic levels of hormones such as insulin-like growth factor-1 (IGF-1), leptin and insulin that mediate energy balance. Dual-energy X-ray absorptiometry showed that C/EBPβ, either directly or indirectly, modulated body weight, fat content and bone density in both males and females, while the effect of C/EBP was minor and only affected adiposity and body weight in female animals. Levels of IGF-1, leptin and insulin in the serum were decreased in both male and female C/EBPβ^–/– mice, and C/EBPβ was associated with their promoters in vivo. Moreover, colon adenocarcinoma cells displayed reduced tumorigenic potential when transplanted into C/EBPβ-deficient animals, especially males. Thus, C/EBPβ contributes to endocrine expression of IGF-1, leptin and insulin, which modulate energy balance and can contribute to cancer progression by creating a favorable environment for tumor cell proliferation and survival.

CCDC62/ERAP75 functions as a coactivator to enhance estrogen receptor beta-mediated transactivation and target gene expression in prostate cancer cells

Chen, M., Ni, J., Chang, H.-C., Lin, C.-Y., Muyan, M., Yeh, S. — 2009-04-30

Human prostate cancer (PCa) and prostate epithelial cells predominantly express estrogen receptor (ER) β, but not ER. ERβ might utilize various ER coregulators to mediate the E2-signaling pathway in PCa. Here, we identified coiled-coil domain containing 62 (CCDC62)/ERAP75 as a novel ER coactivator. CCDC62/ERAP75 is widely expressed in PCa cell lines and has low expression in MCF7 cells. Both in vitro and in vivo interaction assays using mammalian two-hybrid, glutathione S-transferase pull-down and coimmunoprecipitation methods proved that ERβ can interact with the C-terminus of CCDC62/ERAP75 via the ligand-binding domain. The first LXXLL motif within CCDC62/ERAP75 is required for the interaction between ERβ and CCDC62/ERAP75. Electrophoretic mobility shift assay showed that CCDC62/ERAP75 can be recruited by the estrogen response element–ER complex in the presence of ligand. Furthermore, a chromatin immunoprecipitation assay demonstrated the hormone-dependent recruitment of CCDC62/ERAP75 within the promoter of the estrogen-responsive gene cyclin D1. In addition, using silencing RNA (siRNA) against endogeneous CCDC62/ERAP75, we demonstrated that inhibition of endogenous CCDC62/ERAP75 results in the suppression of ERβ-mediated transactivation as well as target gene expression in LNCaP cells. More importantly, using the tet-on overexpression system, we showed that induced expression of CCDC62/ERAP75 can enhance the E2-regulated cyclin D1 expression and cell growth in LNCaP cells. Together, our results revealed the role of CCDC62/ERAP75 as a novel coactivator in PCa cells that can modulate ERβ transactivation and receptor function.

Interferon-{alpha} counteracts the angiogenic switch and reduces tumor cell proliferation in a spontaneous model of prostatic cancer

2009-04-30

Interferon (IFN)- is a cytokine with marked therapeutic activity in transplantable tumor models, that is in part due to angiogenesis inhibition. Aim of this study was to investigate the effects of IFN- during the early phases of tumor development in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. To provide sustained IFN- production, TRAMP mice were injected intraperitoneally with lentiviral vectors. IFN- administration resulted in rapid and protracted upregulation of IFN--regulated genes associated with antiangiogenic and antiproliferative functions in the prostate of TRAMP mice, including guanylate-binding protein 1 (GBP-1), IFI204 and CXCL10-11. These transcriptional changes were accompanied by effects on the tumor vasculature, including significant reduction of intraductal microvessel density and increased pericyte coverage, and marked reduction of tumor cell proliferation, without induction of tumor necrosis. Intriguingly, GBP-1 and myxovirus resistance A, two IFN-regulated proteins, were found expressed in ~40% of human prostate cancer samples analyzed, suggesting expression of endogenous IFN-. Overall, these findings demonstrate that IFN- is able to counteract the angiogenic switch and impairs tumor cell proliferation in preinvasive lesions. Since the angiogenic switch also marks progression of human prostatic cancer, these results highlight the potential of angiogenesis inhibitors for the development of chemoprevention strategies in high-risk individuals.

Proepithelin is an autocrine growth factor for bladder cancer

2009-04-30

The growth factor proepithelin functions as an important regulator of proliferation and motility. Proepithelin is overexpressed in a great variety of cancer cell lines and clinical specimens of breast, ovarian and renal cancer, as well as glioblastomas. Using recombinant proepithelin on 5637 transitional cell carcinoma-derived cells, we have shown previously that proepithelin plays a critical role in bladder cancer by promoting motility of bladder cancer cells. In this study, we used the ONCOMINE database and gene microarray analysis tool to analyze proepithelin expression in several bladder cancer microarray studies. We found a statistically significant increase in proepithelin messenger RNA expression in bladder cancers vis-à-vis non-neoplastic tissues, and this was associated with pathologic and prognostic parameters. Targeted downregulation of proepithelin in T24 transitional carcinoma cells with small hairpin RNA inhibited both Akt and mitogen-activated protein kinase pathways, severely reduced the ability of T24 cells to proliferate in the absence of serum and inhibited migration, invasion and wound healing. In support of these in vitro results, we discovered that proepithelin expression was significantly upregulated in invasive bladder cancer tissues compared with normal urothelium. In addition, proepithelin was secreted in the urine, where it was detectable by immunoblotting and enzyme-linked immunosorbent assay. Collectively, these results support the hypothesis that proepithelin may play a critical role as an autocrine growth factor in the establishment and progression of bladder cancer and suggest that proepithelin may prove a novel biomarker for the diagnosis and prognosis of bladder neoplasms.

Dysregulation of WNT/CTNNB1 and PI3K/AKT signaling in testicular stromal cells causes granulosa cell tumor of the testis

Boyer, A., Paquet, M., Lague, M.-N., Hermo, L., Boerboom, D. — 2009-04-30

Synergistic effects of dysregulation of the WNT/CTNNB1 and phosphatidylinositol 3-kinase (PI3K)/AKT pathways are thought to be important for the development and progression of many forms of cancer, including the granulosa cell tumor of the ovary. Sustained WNT/CTNNB1 signaling in Sertoli cells causes testicular degeneration and the formation of foci of poorly differentiated stromal cells in the seminiferous tubules in mice. To test if concomitant dysregulation of the WNT/CTNNB1 and PI3K/AKT pathways could synergize to cause testicular cancer, Pten^{tm1Hwu/tm1Hwu};Ctnnb1^tm1Mmt/+;Amhr2^{tm3(cre)Bhr/+} mice that express a dominant, stable CTNNB1 mutant and lack the expression of phosphatase and tensin homolog (PTEN) in their Sertoli cells were generated. These mice developed aggressive testicular cancer with 100% penetrance by 5 weeks of age, and 44% of animals developed pulmonary metastases by 4 months, whereas Pten^{tm1Hwu/tm1Hwu};Amhr2^{tm3(cre)Bhr/+} controls were phenotypically normal. Surprisingly, the tumors could not be classified as Sertoli cell tumors, but rather bore histologic and ultrastructural characteristics of granulosa cell tumors of the testis (GCTT). Pten^{tm1Hwu/tm1Hwu};Ctnnb1^tm1Mmt/+;Amhr2^{tm3(cre)Bhr/+} testicular tumors did not express CYP17, CYP19, germ cell nuclear antigen, estrogen receptor 1 or progesterone receptor, but expressed the early granulosa cell markers WNT4 and FOXL2, confirming the diagnosis of GCTT. Immunohistochemical analyses of Pten^{tm1Hwu/tm1Hwu};Ctnnb1^tm1Mmt/+;Amhr2^{tm3(cre)Bhr/+} GCTT demonstrated a tumor marker profile similar to that reported in human GCTT. Immunoblotting analyses revealed high levels of phosphorylation of AKT and the PI3K/AKT signaling effector FOXO1A in Pten^{tm1Hwu/tm1Hwu};Ctnnb1^tm1Mmt/+;Amhr2^{tm3(cre)Bhr/+} GCTT, suggesting the involvement of FOXO1A in the mechanism of GCTT development. Together, these data provide the first insights into the molecular etiology of GCTT and the first animal model for the study of GCTT biology.

Predominant modifier of extreme liver cancer susceptibility in C57BR/cdJ female mice localized to 6 Mb on chromosome 17

Peychal, S. E.-M., Bilger, A., Pitot, H. C., Drinkwater, N. R. — 2009-04-30

Sex hormones influence the susceptibility of inbred mice to liver cancer. C57BR/cdJ (BR) females are extremely susceptible to spontaneous and chemically induced liver tumors, in part due to a lack of protection against hepatocarcinogenesis normally offered by ovarian hormones. BR males are also moderately susceptible, and the susceptibility of both sexes of BR mice to liver tumors induced with N,N-diethylnitrosamine relative to the resistant C57BL/6J (B6) strain is caused by two loci designated Hcf1 and Hcf2 (hepatocarcinogenesis in females) located on chromosomes 17 and 1, respectively. The Hcf1 locus on chromosome 17 is the predominant modifier of liver cancer in BR mice. To validate the existence of this locus and investigate its potential interaction with Hcf2, congenic mice for each region were generated. Homozygosity for the B6.BR(D17Mit164-D17Mit2) region resulted in a 4-fold increase in liver tumor multiplicity in females and a 4.5-fold increase in males compared with B6 controls. A series of 16 recombinants covering the entire congenic region was developed to further narrow the area containing Hcf1. Susceptible heterozygous recombinants demonstrated a 3- to 7-fold effect in females and a 1.5- to 2-fold effect in males compared with B6 siblings. The effect in susceptible lines completely recapitulated the susceptibility of heterozygous full-length chromosome 17 congenics and furthermore narrowed the location of the Hcf1 locus to a single region of the chromosome from 30.05 to 35.83 Mb.

Incorporation of 5-chlorocytosine into mammalian DNA results in heritable gene silencing and altered cytosine methylation patterns

Lao, V. V., Herring, J. L., Kim, C. H., Darwanto, A., Soto, U., Sowers, L. C. — 2009-04-30

Cytosine methylation patterns are essential for the proper control of gene expression in higher vertebrates. Although alterations in methylation patterns are frequently observed in human tumors, neither the mechanisms for establishing methylation patterns during normal development nor the mechanisms leading to pathological alterations of methylation patterns are currently known. While epidemiological studies have implicated inflammation in cancer etiology, a mechanistic link has yet to be established. Investigations of inflammation-mediated DNA damage may have provided important new insights. Our in vitro studies revealed that the inflammation-mediated DNA damage product, 5-chlorocytosine, could direct fraudulent methylation of previously unmethylated CpG sites. The purpose of this study was to recapitulate our in vitro findings by introducing 5-chlorocytosine residues into the DNA of replicating mammalian cells and to examine its impact on gene expression and cytosine methylation patterns. CHO-K1 cells hemizygous for the hprt gene were electroporated with the triphosphates of cytosine [2'-deoxycytidine-5'-triphosphate (dCTP)], 5-methylcytosine [5-methyl-2'-deoxycytidine-5'-triphosphate (MedCTP)] and 5'-chloro-2'-deoxycytidine-5'-triphosphate (CldCTP), and then selected with 6-thioguanine for silencing the hprt gene. Both modified nucleotides, MedCTP and CldCTP, but not unmodified dCTP, silenced hprt gene expression. Subsequent bisulfite pyrosequencing of CpG sites within the hprt promoter region of the selected cells confirmed hypermethylation, although global methylation levels as measured by gas chromatography–mass spectrometry did not change. Modified nucleotide-induced gene silencing could be reversed with 5-aza-2'-deoxycytidine indicating an epigenetic rather than mutagenic alteration. These results provide further evidence that the inflammation damage product 5-chlorocytosine could be a link between inflammation and cancer development.

Overexpression of astrocyte elevated gene-1 (AEG-1) is associated with esophageal squamous cell carcinoma (ESCC) progression and pathogenesis

2009-04-30

Astrocyte elevated gene-1 (AEG-1), upregulated in various types of human cancers, has been reported to be associated with the carcinogenesis of human cancer. However, the functional significance of AEG-1 in human esophageal squamous cell carcinoma (ESCC) remains unknown. In the present study, we showed the expression of AEG-1 was markedly upregulated in esophageal cancer cell lines and surgical ESCC specimens at both transcriptional and translational levels. Immunohistochemical analysis revealed that 80 of 168 (47.6%) paraffin-embedded archival ESCC specimens exhibited high levels of AEG-1 expression. Statistical analysis suggested the upregulation of AEG-1 was significantly correlated with the clinical staging of the ESCC patients (P = 0.001), T classification (P = 0.002), N classification (P = 0.034), M classification (P = 0.021) and histological differentiation (P = 0.035) and those patients with high AEG-1 levels exhibited shorter survival time (P < 0.001). Multivariate analysis indicated that AEG-1 expression might be an independent prognostic indicator of the survival of patients with ESCC. Furthermore, we found that ectopic expression of AEG-1 in ESCC cells could significantly enhance cell proliferation and anchorage-independent growth ability. Conversely, silencing AEG-1 by short hairpin RNAi caused an inhibition of cell growth and anchorage-independent growth ability on soft agar. Moreover, we demonstrated that the upregulation of AEG-1 could reduce the expression of p27^Kip1 and induce the expression of cyclin D1 through the AKT/FOXO3a pathway. Our findings suggest that the AEG-1 protein is a valuable marker of ESCC progression and that the upregulation of AEG-1 plays an important role in the development and pathogenesis of human ESCC.