Carcinogenesis - Advance Access

Activation of JAK2/STAT3 signaling by osteopontin promotes tumor growth in human breast cancer cells

Behera, R., Kumar, V., Lohite, K., Karnik, S., Kundu, G. C. — Thu, 19 Nov 2009 00:03:55 PST

Deregulation of STAT3 signaling plays crucial role in oncogenesis of various cancers. However, the molecular mechanism by which osteopontin (OPN), a chemokine like ECM associated protein regulates STAT3 activation which leads to tumor progression and inhibits apoptosis in breast cancer cells is not well understood. In this study, we for the first time report that OPN upregulates vβ3 integrin-mediated JAK2 phosphorylation and STAT3 activation in breast cancer (MDA-MB-468 and MCF-7) cells. Pretreatment of cells with JAK2 inhibitor (AG 490) suppresses OPN-induced STAT3 phosphorylation, its nuclear localization and DNA-binding indicating that JAK2 is involved in this process. Transfection of cells with wild type STAT3 (wt STAT3) enhanced whereas mutant STAT3 (STAT3 Y705F) suppressed OPN-induced breast tumor cell migration. Treatment of cells with OPN followed by staurosporine showed that OPN protects the cells from staurosporine-induced apoptosis. Moreover, transfection of cells with wt STAT3 upregulates whereas STAT3 Y705F downregulates Bcl2 and cyclin D1 expressions in response to OPN. Interestingly, STAT3 overexpressing cells when injected to NOD/SCID mice followed by OPN treatment, the mice developed enhanced tumor growth as compared to STAT3 Y705F injected mice or mice injected with OPN alone. The levels of Bcl2 and cyclin D1 in wt STAT3 tumors were significantly higher than controls. Clinical specimen analysis revealed that increased OPN and pSTAT3 expressions correlate with enhanced breast tumor progression. Thus, targeting OPN and its regulated STAT3 signaling could be a potent therapeutic approach and understanding these mechanisms may form the basis of new therapeutic regimen for the management of breast cancer.

Oral administration of caffeine during voluntary exercise markedly decreases tissue fat and stimulates apoptosis and cyclin B1 in UVB-treated skin of hairless p53 knockout mice

Lou, Y., Peng, Q., Nolan, B., Wagner, G. C., Lu, Y. — Thu, 19 Nov 2009 00:03:54 PST

Treatment of p53(-/-) mice orally with caffeine, voluntary exercise, or their combination for 2 weeks prior to a single irradiation with UVB (a) decreased the weight of the epididymal fat pads by 22, 40 and 56%, (b) decreased the thickness of the dermal fat layer by 10, 26, and 42%, (c) increased the number of apoptotic sunburn cells by 29, 100 and 489%, (d) increased the number of caspase 3 positive cells by 33, 117 and 667%, and (e) increased the number of mitotic cells with cyclin B1-positive staining by 40, 210 and 510%, respectively. Pearson's correlation coefficient indicated a statistically significant inverse relationship between the level of tissue fat and the number of mitotic cells with cyclin B1 in p53(-/-) mice but not in p53(+/+) littermates. Western blot analysis indicated that treatment of p53(-/-) mice with caffeine together with exercise increased the level of cyclin B1 significantly more than in p53(+/+) mice. p53(-/-) mice, but not p53(+/+) mice, treated with caffeine during exercise exhibited a dramatic decrease in the level of survivin. Our results suggest that voluntary exercise in combination with oral caffeine may exert a synergistic increase in UVB-induced apoptosis and that tissue fat may be a more important modulator of apoptosis and carcinogenesis in p53 deficient mice than in p53 normal mice. The stimulatory effects on apoptosis in p53(-/-) mice by the combination treatment might be associated with increased levels of cyclin B1 and decreased levels of survivin.

Non-codingRNA Sequence Variations in Human Chronic Lymphocytic Leukemia and Colorectal Cancer

Thu, 19 Nov 2009 00:03:54 PST

Cancer is a genetic disease in which the interplay between alterations in protein coding genes and non-coding RNAs (ncRNAs) plays a fundamental role. In recent years, the full coding component of the human genome was sequenced in various cancers, while such attempts related to ncRNAs are still fragmentary. We screened genomic DNAs for sequence variations in 148 microRNAs (miRNAs) and ultraconserved regions (UCRs) loci in patients with chronic lymphocytic leukemia (CLL) or colorectal cancer (CRC) by Sanger technique and further tried to elucidate the functional consequences of some of these variations. We found sequence variations in miRNAs in both sporadic and familial CLL cases, mutations of UCRs in CLLs and CRCs and, in certain instances, detected functional effects of these variations. Furthermore, by integrating our data with previously published data on miRNA sequence variations, we have created a catalog of DNA sequence variations in miRNAs/UCGs in human cancers. These findings argue that ncRNAs are targeted by both germ-line and somatic mutations as well as by single-nucleotide polymorphisms with functional significance for human tumorigenesis. Sequence variations in ncRNA loci are frequent and some have functional and biological significance. Such information can be exploited to further investigate on a genome-wide scale the frequency of genetic variations in ncRNAs and their functional meaning, as well as for the development of new diagnostic and prognostic markers for leukemias and carcinomas.

Oncofetal H19-derived miR-675 Regulates Tumor Suppressor RB in Human Colorectal Cancer

Thu, 19 Nov 2009 00:03:53 PST

H19 is an imprinted oncofetal non-coding RNA recently shown to be the precursor of miR-675. The pathophysiologic roles of H19 and its mature product miR-675 to carcinogenesis have however not been defined. By quantitative RT-PCR, both H19 and miR-675 were found to be up-regulated in human colon cancer cell lines and primary human colorectal cancer (CRC) tissues compared with adjacent non-cancerous tissues. Subsequently, the tumor suppressor RB was confirmed to be a direct target of miR-675 as the miRNA suppressed the activity of the luciferase reporter carrying the 3’UTR of RB mRNA that contains the miR-675 binding site. Suppression of miR-675 by transfection with anti-miR-675 increased RB expression, and at the same time, decreased cell growth and soft agar colony formation in human colon cancer cells. Reciprocally, enhanced miR-675 expression by transfection with miR-675 precursor decreased RB expression, increased tumor cell growth and soft agar colony formation. Moreover, the inverse relationship between the expressions of RB and H19/miR-675 was also revealed in human CRC tissues and colon cancer cell lines. Our findings demonstrate that H19-derived miR-675, through down-regulation of its target RB, regulates the CRC development and thus may serve as a potential target for CRC therapy.

An insertion/deletion polymorphism at miRNA-122 binding site in the interleukin-1{alpha} 3' untranslated region confers risk for hepatocellular carcinoma

Mon, 16 Nov 2009 06:53:01 PST

Hepatocellular carcinoma (HCC) is the fifth most common malignancy caused by environmental and genetic factors. MicroRNAs (miRNAs) are a class of short non-coding RNAs with posttranscriptional regulatory functions. They participate in diverse biological pathways and function as gene regulators. Genetic polymorphisms in 3’ untranslated regions (3'UTR) targeted by miRNAs alter the strength of miRNA binding, with consequences on regulation of target genes thereby affecting the individual's cancer risk. We have previously predicted polymorphisms falling in microRNA-binding regions of cancer genes. We selected an insertion/deletion (Indel) polymorphism (rs3783553) in the 3'untranslated region of interleukin-1 (IL1A) for a case-control study in a Chinese population. With samples from 403 HCC patients and 434 healthy control individuals, strong evidence of association was observed for the variant homozygote. This association was validated in a second independent case-control study with 1074 HCC patients and 1239 healthy control individuals (OR = 0.62 95%CI = 0.49-0.78). We further show that the "TTCA" insertion allele for rs3783553 disrupts a binding site for microRNA-122 and microRNA-378, thereby increasing transcription of interleukin-1 (IL-1) in vitro and in vivo. These findings suggest that functional polymorphism rs3783553 in IL1A could contribute to HCC susceptibility. Considering IL-1 affects not only various phases of the malignant process, such as carcinogenesis, tumor growth, and invasiveness, but also patterns of interactions between malignant cells and the host's immune system, our results indicated that IL-1 may be a promising target for immunotherapy, early diagnosis and intervention of HCC.

Effects of HPV-16 E5, E6 and E7 Proteins on Survival, Adhesion, Migration and Invasion of Trophoblastic Cells

Mon, 16 Nov 2009 06:53:01 PST

Amongst high-risk human papillomaviruses (HPV), HPV-16 infection is the most prevalent causative factor for cervical cancer. Beside other mucosal targets, HPV-16 was reported to infect the placenta and to replicate in trophoblastic cells. Since these cells share invasive properties of tumoral cells, they represent an ideal model to investigate several oncogenic processes. In the present work, we analyzed the impacts of HPV-16 E5, E6 and E7 oncoproteins on the trophoblastic model. Our results showed that E5 impaired the viability of trophoblastic and cervical cell lines but E6 and E7, favouring cell growth, neutralised the E5 cytotoxic effect. In addition, E5 decreased the adhesiveness of trophoblastic cells to the tissue culture plastic and to endometrial cells similarly as previously described for E6 and E7. E5 and E6 plus E7 increased also their migration and their invasive properties. Cells expressing HPV-16 early proteins under the control of the LCR endogenous promoter displayed growth advantage and were also more motile and invasive compared to control cells. Interestingly, the E-cadherin was down regulated in trophoblastic cells expressing E5, E6 and E7. NF-B and AP-1 activities were also enhanced. In conclusion, HPV-16 early proteins enhanced trophoblastic growth and intensify the malignant phenotype by impairing cell adhesion leading to increased cellular motile and invasive properties. HPV-16 E5 participated, with E6 and E7, in these changes by impairing E-cadherin expression, a hallmark of malignant progression.

Dual Promoter Regulation of Death-associated Protein Kinase Gene Leads to Differentially Silenced Transcripts by Methylation in Cancer

Mon, 16 Nov 2009 06:53:00 PST

Death associated protein kinase (DAPK), a mediator of apoptotic systems, is silenced by promoter hypermethylation in lung and breast tumors. This gene has a CpG island extending 2500 bp from the translational start site; however, studies characterizing its transcriptional regulation have not been conducted. Two transcripts for DAPK were identified that code for a single protein, while being regulated by two promoters. The previously identified DAPK transcript designated exon 1 transcript was expressed at levels 3-fold greater than the alternate exon 1b transcript. Deletion constructs of promoter 1 identified a 332 bp region containing a functional CP2 binding site important for expression of the exon 1 transcript. While moderate reporter activity was seen in promoter 2, the region comprising intron 1 and containing a HNF3B binding site sustained expression of the alternate transcript. Sequencing the DAPK CpG island in tumor cell lines revealed dense, but heterogeneous methylation of CpGs that blocked access of the CP2 and HNF3B proteins that in turn, was associated with loss of transcription that was restored by treatment with 5-aza-2'-deoxycytidine. Prevalences were similar for methylation of promoter 1 and 2 and intron 1 in lung tumors, but significantly greater in promoter 2 and intron 1 in breast tumors, indicative of tissue-specific differences in silencing these two transcripts. These studies show for the first time, dual promoter regulation of DAPK, a tumor suppressor gene silenced in many cancers, and substantiate the importance of screening for silencing of both transcripts in tumors.

Nicotine dependence may link the 15q25 locus to lung cancer risk

Galvan, A., Dragani, T. A. — Thu, 12 Nov 2009 02:07:28 PST

The nicotinic 15q25 locus has been implicated in lung cancer risk, with an odds ratio of ~1.3. The same locus is associated with nicotine dependence due to cigarette smoking and with smoking-associated chronic obstructive pulmonary disease, which is a risk factor for lung cancer. Our meta-analysis of reported studies shows that this locus was not associated with lung cancer risk in >1000 never-smoker cases and >1800 controls. Review of exposure-response data for lung cancer risk showed that less than a half-cigarette/day may confer the same risk of lung cancer as that conferred by the 15q25 locus. Given the lack of effect in never-smokers and the known common and variable underreporting of smoking habit in studies on smoking-associated diseases, we cannot exclude that the association between the 15q25 locus and lung cancer risk is indirect, deriving from association of the same locus with smoking habit. Since nicotine is not carcinogenic, available data do not provide plausibility of the association between the nicotinic 15q25 locus and lung cancer pathogenesis. Thus, a direct link between the 15q25 locus and lung cancer risk has yet to be established.

Genome-wide Association Studies in Cancer - Current and Future Directions

Chung, C. C., Magalhaes, W., Gonzalez-Bosquet, J., Chanock, S. J. — Wed, 11 Nov 2009 05:41:46 PST

Genome-wide association studies (GWAS) have emerged as an important tool for discovering regions of the genome that harbor genetic variants that confer risk for different types of cancers. The success of GWAS in the last five years is due to the convergence of new technologies that can genotype hundreds of thousands of single nucleotide polymorphism (SNP) markers together with comprehensive annotation of genetic variation. This approach has provided the opportunity to scan across the genome in a sufficiently set of cases and controls without a set of prior hypotheses in search of susceptibility alleles with low effect sizes. Generally, the susceptibility alleles discovered thus far are common, namely, with a frequency in one or more population of greater than 5% and each allele confers a small contribution to the overall risk for the disease. For nearly all regions conclusively identified by GWAS, the per allele effect sizes estimated are less than 1.3. Consequently, the findings of GWAS underscore the complex nature of cancer and have focused attention on a subset of the genetic variants that comprise the genomic architecture of each type of cancer, which already we can see can differ substantially by the number of regions associated with specific types of cancer. For instance, in prostate cancer, there could be more than 30 distinct regions harboring common susceptibility alleles identified by GWAS whereas in lung cancer, a disease strongly driven by exposure to tobacco products, so far, only three regions have been conclusively established. To date, over 85 regions have been conclusively associated in over a dozen different cancers, yet no more than five regions have been associated with more than one distinct cancer type. GWAS are an important discovery tool that require extensive follow-up to map each region, investigate the biological mechanism underpinning the association and eventually test the optimal markers for assessing risk for a disease or its outcome, such as in pharmacogenomics, the study of the effect of genetic variation on pharmacological interventions. The success of GWAS has opened new horizons for exploration and highlighted the complex genomic architecture of disease susceptibility.

MicroRNA Expression in Head and Neck Cancer Associates with Alcohol Consumption and Survival

Avissar, M., McClean, M. D., Kelsey, K. T., Marsit, C. J. — Mon, 09 Nov 2009 04:00:32 PST

The contribution of microRNAs (miRNAs) to carcinogenesis in many tumors, including head and neck squamous cell carcinomas (HNSCCs) is clear, but the etiology and clinical significance of their alteration remain important questions. Our previous work has identified four microRNAs (miRNAs) as differentially expressed HNSCCs compared to non-diseased epithelia and showed that there is potential diagnostic utility in examining their expression. Here, we used quantitative real-time PCR to determine the relative expression of these miRNAs in a larger, independent case-series of HNSCC tumors (n = 169), examining associations of miRNA expression with exposures and clinical features associated with HNSCC. In multivariate analyses, expression of miR-375 was shown to increase with alcohol consumption (P = 0.002), and showed higher expression in tumors of pharyngeal and laryngeal origin compared with oral tumors (P < 0.05 and P < 0.01, respectively). Additionally, high miR-21 expression was associated with significantly decreased 5-year survival in patients (HR, 1.68; 95% CI 1.04-2.77) in a model controlled for patient age, gender, and tumor stage. Together, these data suggest that alterations in miRNA expression are related to exposures causal in head and neck cancer and may be useful biomarkers of patient outcome.

The Properties of Tumor-initiating Cells from a Hepatocellular Carcinoma Patient's Primary and Recurrent Tumor

Fri, 06 Nov 2009 07:17:58 PST

Hepatocellular carcinoma (HCC) is associated with a high morbidity and mortality due to its high rate of recurrence. However, little is known about the biological characteristics of recurrent HCC cells. A single patient's primary and recurrent HCC derived cell lines, Hep-11 and Hep-12 respectively, were established by primary culture. These two cell lines have the same HBV integration site and share many common amplifications and deletions, which suggest that they have the same clonal origin. While Hep-11 cells were non-tumorigenic at 16 weeks following injection of up to 10,000 cells, injection of only 100 Hep-12 cells was sufficient to initiate tumor growth, and all single Hep-12 clones were tumorigenic in immunodeficient mice. Compared with Hep-11, Hep-12 cells expressed the oval cell markers AFP, NCAM/CD56, c-kit/CD117, as well as multiple stem cell markers such as Nanog, OCT4, and SOX2. In addition, greater than 90% of Hep-12 cells were aldehyde dehydrogenase positive. They were also less resistant to paclitaxel, but more resistant to doxorubicin, cisplatin and hydroxycamptothecin (HCPT) which had been administrated to the patient. Furthermore, Hep-12 expressed higher levels of poly (ADP-ribose) polymerase-1 (PARP-1) than Hep-11, and PARP-1 inhibition potentiated the sensitivity to HCPT in Hep-12 but not in Hep-11 cells. These results indicate that a large population of the recurrent HCC-derived Hep-12 cells were tumor-initiating cells, and that elevated expression of PARP-1 was related to their resistance to HCPT.

Shorter telomere length in peripheral blood lymphocytes of workers exposed to polycyclic aromatic hydrocarbons

Thu, 05 Nov 2009 05:52:03 PST

Shorter telomere length in peripheral blood lymphocytes (PBLs) is predictive of lung cancer risk. Polycyclic aromatic hydrocarbons (PAHs) are established lung carcinogens that cause chromosome instability. Whether PAH exposure and its molecular effects are linked with shorter telomere length has never been evaluated. In the present study, we investigated the effect of chronic exposure to PAHs on telomere length measured in PBLs of Polish male non-current smoking cokeoven workers and matched controls. PAH exposure and molecular effects were characterized using measures of internal dose (urinary 1-pyrenol), effective dose (anti-BPDE-DNA adduct), genetic instability (micronuclei, MN), and DNA methylation (p53 promoter, and Alu and LINE-1 repetitive elements, as surrogate measures of global methylation) in PBLs. Telomere length was measured by real-time PCR.

Cokeoven workers were heavily exposed to PAHs (79% exceeded the urinary 1-pyrenol biological exposure index), and exhibited lower telomere length(p = 0.038) than controls, as well as higher levels of genetic and chromosomal alterations (i.e., anti-BPDE-DNA adduct and MN [p<0.0001]), and epigenetic changes (i.e., p53 gene-specific promoter and global methylation [p≤0.001]). Telomere length decreased with longer duration of work as cokeoven worker(p = 0.039) and in all subjects with higher levels of anti-BPDE-DNA adduct(p = 0.042), p53 hypomethylation(p = 0.005), and MN(p = 0.009). In multivariate analysis, years of work in cokery(p = 0.008) and p53 hypomethylation(p = 0.001) were the principal determinants of shorter telomere length.

Our results indicate that shorter telomere length is associated with chronic PAH exposure. The interrelations with other genetic and epigenetic mechanisms in our data suggests that shorter telomere length could be a central event in PAH carcinogenesis.

Genetic Polymorphisms in the PTPN13 gene and Risk of Squamous Cell Carcinoma of Head and Neck

Niu, J., Huang, Y.-J., Wang, L.-E, Sturgis, E. M., Wei, Q. — Thu, 05 Nov 2009 05:52:02 PST

Fas-associated phosphatase 1 (FAP-1) is encoded by the protein tyrosine phosphatase, non-receptor type 13 (PTPN13) gene and attributes to the resistance to Fas-mediated apoptosis in several tumors, including squamous cell carcinoma of head and neck (SCCHN). However, no epidemiological studies have investigated the roles of PTPN13 polymorphisms in SCCHN risk. In this hospital based case-control study of 1069 SCCHN patients and 1102 non-Hispanic white cancer-free controls, we evaluated the associations between three single nucleotide polymorphisms (SNPs) c.4068 T>G F1356L (rs10033029), c.4566 A>G I1522M (rs2230600) and c.6241 T>G Y2081D (rs989902) located in the coding region of PTPN13 and SCCHN risk. We found that a significantly increased SCCHN risk was associated with c.4566 I1522M GG genotype (OR = 1.89, 95% CI = 1.27-2.79) and c.6241 Y2081D GT genotype (OR = 1.26, 95% CI = 1.03-1.53) compared with the c.4566 I1522M AA and c.6241 Y2081D TT genotypes, respectively. Further stratified analyses showed that risk associated with c.4566 I1522M GG genotype was more profound in the subgroups of young (≤57 years), males, never smokers, current drinkers, and patients with pharyngeal cancer; that risk associated with c.6241 Y2081D GT genotype persisted in subgroups of old (>57 years), males, current drinkers, and patients with pharyngeal and laryngeal cancers; and that risk associated with c.6241 Y2081D GG genotype was borderline in patients with laryngeal cancer. In conclusion, polymorphisms in the PTPN13 coding region may be biomarkers for susceptibility to SCCHN in US populations.

MMP13 as a stromal mediator in controlling persistent angiogenesis in skin carcinoma

Thu, 05 Nov 2009 05:52:01 PST

Matrix metalloproteinases (MMPs) such as MMP13 promote tumour growth and progression by mediating ECM reorganization and regulating the biological activity of cytokines. Using Mmp13-/- mice, we demonstrate an essential role of this single collagenase for highly malignant and invasive growth in skin squamous cell carcinoma (SCC). Lack of host MMP13 strongly impaired tumour growth of malignant SCC cells, leading to small, mostly avascular cysts. While initial stromal activation in tumour transplants of Mmp13+/+ and Mmp13-/- animals was similar, MMP13 was essential for maintenance of angiogenesis and for invasion. MMP13 was induced in fibroblasts of the wildtype animals at the onset of invasion and correlated with a strong increase in VEGF protein and its association with VEGFR-2 on endothelial cells in invasive areas. In contrast, VEGF protein in the stroma was barely detectable and tumour invasion was downregulated in Mmp13-/- animals, despite ongoing VEGF mRNA expression. Taken together with in vitro data showing the release of VEGF from the ECM by MMP13 expressing fibroblasts, these data strongly suggest a crucial role of MMP13 in promoting angiogenesis via releasing VEGF from the ECM and thus allowing the invasive growth of the SCC cells.

Senescence: an antiviral defense that is tumor suppressive?

Reddel, R. R. — Tue, 03 Nov 2009 05:10:51 PST

Normal mammalian somatic cells proliferate a finite number of times in vitro before permanently withdrawing from the cell cycle into a cellular state referred to as senescence. Senescence may be triggered by excessive mitogenic stimulation or by various forms of cellular damage including excessive telomere shortening. Over the past decade, there has been continuing accumulation of evidence that senescence occurs in vivo, that it is relevant to aging, and that it has a tumor suppressor function. However, it has also become clear that the phenotype of senescence also has a number of puzzling features, including the secretion of pro-inflammatory factors that may foster tumorigenesis and also the senescence of neighboring cells. On the basis of these antagonistic pro- and anti-tumorigenic effects, and of the observation that many viruses have developed proteins that prevent senescence of the cells they infect, it is argued that the primary function of senescence may have been as an antiviral defense mechanism. Recent progress in understanding how tumor cells evade senescence is also reviewed here.

Telomeres and telomerase in cancer

Artandi, S. E., DePinho, R. A. — Tue, 03 Nov 2009 05:10:51 PST

Monocyclic Aromatic Amines as Potential Human Carcinogens: Old is New Again

Skipper, P. L., Kim, M. Y., Patty Sun, H.-L., Wogan, G. N., Tannenbaum, S. R. — Tue, 03 Nov 2009 15:46:04 PST

Alkylanilines are a group of chemicals whose ubiquitous presence in the environment is a result of the multitude of sources from which they originate. Exposure assessments indicate that most individuals experience lifelong exposure to these compounds. Many alkylanilines have biological activity similar to that of the carcinogenic multi-ring aromatic amines. This review provides an overview of human exposure and biological effects. It also describes recent investigations into the biochemical mechanisms of action that lead to the assessment that they are most likely more complex than those of the more extensively investigated multi-ring aromatic amines. Not only is nitrenium ion chemistry implicated in DNA damage by alkylanilines, but also reactions involving quinone imines and perhaps reactive oxygen species. Recent results described here indicate that alkylanilines can be potent genotoxins for cultured mammalian cells when activated by exogenous or endogenous phase I and phase II xenobiotic metabolizing enzymes. The nature of specific DNA damage products responsible for mutagenicity remains to be identified but evidence to date supports mechanisms of activation through obligatory N-hydroxylation as well as subsequent conjugation by sulfation and/or acetylation. A fuller understanding of the mechanisms of alkylaniline genotoxicity is expected to provide important insights into the environmental and genetic origins of one or more human cancers and may reveal a substantial role for this group of compounds as potential human chemical carcinogens.

Mycotoxins and human disease: a largely ignored global health issue

Wild, C. P., Gong, Y. Y. — Thu, 29 Oct 2009 12:11:01 PDT

Aflatoxins and fumonisins are mycotoxins contaminating a large fraction of the world's food, including maize, cereals, groundnuts and tree nuts. The toxins frequently co-occur in maize. Where these commodities are dietary staples, for example in parts of Africa, Asia and Latin America, the contamination translates to high level, chronic exposure. This is particularly true in subsistence farming communities where regulations to control exposure are either non-existent or practically unenforceable. Aflatoxins are hepatocarcinogenic in humans, particularly in conjunction with chronic hepatitis B virus infection, and cause aflatoxicosis in episodic poisoning outbreaks. In animals these toxins also impair growth and are immunosuppressive; the latter effects are of increasing interest in human populations. Fumonisins have been reported to induce liver and kidney tumours in rodents and are classified as Group 2B "possibly carcinogenic to humans", with ecological studies implying a possible link to increased oesophageal cancer. Recent studies also suggest the fumonisins may cause neural tube defects in some maize-consuming populations. There is a plausible mechanism for this effect via a disruption of ceramide synthase and sphingolipid biosynthesis. Notwithstanding the need for a better evidence-base on mycotoxins and human health, supported by better biomarkers of exposure and effect in epidemiological studies, the existing data are sufficient to prioritize exposure reduction in vulnerable populations. For both toxins there are a number of practical primary and secondary prevention strategies which could be beneficial if the political will and financial investment can be applied to what remains a largely and rather shamefully ignored global health issue.

Chemical Biology of Mutagenesis and DNA Repair: Cellular Responses to DNA Alkylation

Shrivastav, N., Li, D., Essigmann, J. M. — Thu, 29 Oct 2009 12:11:00 PDT

The reaction of DNA damaging agents with the genome results in a plethora of lesions, commonly referred to as adducts. Adducts may cause DNA to mutate, they may represent the chemical precursors of lethal events, and they can disrupt expression of genes. Determination of which adduct is responsible for each of these biological endpoints is difficult, but this task has been accomplished for some carcinogenic DNA damaging agents. Here, we describe the respective contributions of specific DNA lesions to the biological effects of low molecular weight alkylating agents.

Genetic variations in PI3K-AKT-mTOR pathway and bladder cancer risk

Thu, 29 Oct 2009 12:11:00 PDT

Genetic variations in PI3K-AKT-mTOR pathway may affect critical cellular functions and increase an individual's cancer risk. We systematically evaluate 231 single nucleotide polymorphisms (SNPs) in 19 genes in the PI3K-AKT-mTOR signaling pathway as predictors of bladder cancer risk. In individual SNP analysis, 4 SNPs in RAPTOR remained significant after correcting for multiple testing: rs11653499 (OR: 1.79, 95%CI: 1.24-2.60, P = 0.002), rs7211818 (OR: 2.13, 95%CI: 1.35-3.36, P = 0.001), rs7212142 (OR: 1.57, 95%CI: 1.19-2.07, P = 0.002), and rs9674559 (OR: 2.05, 95%CI: 1.31-3.21, P = 0.002), among which rs7211818 and rs9674559 are within the same haplotype block. In haplotype analysis, compared to the most common haplotypes, haplotype containing the rs7212142 wild type allele showed a protective effect of bladder cancer (OR:0.83, 95%CI: 0.70-0.97). In contrast, the haplotype containing the rs7211818 variant allele showed a 1.32-fold elevated bladder cancer risk (95%CI: 1.09-1.60). In combined analysis of 3 independent significant RAPTOR SNPs (rs11653499, rs7211818, and rs7212142), a significant trend was observed for increased risk with an increase in the number of unfavorable genotypes (P for trend<0.001). Compared to the subjects without any of the unfavorable genotypes, those carrying all 3 unfavorable genotypes showed a 2.22 fold (95%CI: 1.33-3.71) fold increased bladder cancer risk. This is the first study to evaluate the role of germline genetic variations in PI3K-AKT-mTOR pathway as cancer susceptibility factors which will help us identify high risk individuals for bladder cancer.

The human intra-S checkpoint response to UVC-induced DNA damage

Kaufmann, W. K. — Thu, 29 Oct 2009 12:11:02 PDT

The intra-S checkpoint response to 254 nm light (UVC)-induced DNA damage appears to have dual functions to slow the rate of DNA synthesis and stabilize replication forks that become stalled at sites of UVC-induced photoproducts in DNA. These functions should provide more time for repair of damaged DNA before its replication and thereby reduce the frequencies of mutations and chromosomal aberrations in surviving cells. This review tries to summarize the history of discovery of the checkpoint, the current state of understanding of the biological features of intra-S checkpoint signaling, and its mechanisms of action with a focus primarily on intra-S checkpoint responses in human cells. The differences in the intra-S checkpoint responses to UVC- and ionizing-radiation (IR)-induced DNA damage are emphasized. Evidence that [6-4]pyrimidine-pyrimidone (6-4PP) photoproducts in DNA trigger the response are discussed and the relationships between cellular responses to UVC and the molecular dose of UVC-induced DNA damage are briefly summarized. The role of the intra-S checkpoint response in protecting against solar radiation carcinogenesis remains to be determined.

Differential effects of reactive nitrogen species on DNA base excision repair initiated by the alkyladenine DNA glycosylase

Wed, 28 Oct 2009 08:27:45 PDT

Chronic generation of reactive nitrogen species (RNS) can cause DNA damage and may also directly modify DNA repair proteins. RNS-modified DNA is repaired predominantly by the base excision repair (BER) pathway, which includes the alkyladenine DNA glycosylase (AAG). The AAG active site contains several tyrosines and cysteines that are potential sites for modification by RNS. In vitro, we demonstrate that RNS differentially alter AAG activity depending on the site and type of modification. Nitration of tyrosine 162 impaired 1,N⁶-ethenoadenine (A)-excision activity, whereas nitrosation of cysteine 167 increased A-excision. To understand the effects of RNS on BER in vivo, we examined intestinal adenomas for levels of inducible nitric oxide synthase (iNOS) and AAG. A striking correlation between AAG and iNOS expression was observed (r = 0.76, p = 0.00002). Interestingly, there was no correlation between changes in AAG levels and enzymatic activity. We found AAG to be nitrated in human adenomas, suggesting that this RNS modification is relevant in the human disease. Expression of key downstream components of BER, apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase β (POLβ), was also examined. POLβ protein was increased in nearly all adenomas compared to adjacent non-tumor tissues, whereas APE1 expression was only increased in ~half of the adenomas and also was re-localized to the cytoplasm in adenomas. Collectively, the results suggest that BER is dysregulated in colon adenomas. RNS-induced post-translational modification of AAG is one mechanism of BER dysregulation, and the type of modification may define the role of AAG during carcinogenesis.

Cancer Systems Biology: A Network Modeling Perspective

Kreeger, P. K., Lauffenburger, D. A. — Tue, 27 Oct 2009 12:30:56 PDT

Cancer is now appreciated as not only a highly heterogeneous pathology with respect to cell type and tissue origin, but also as a disease involving dysregulation of multiple pathways governing fundamental cell processes such as death, proliferation, differentiation, and migration. Thus, the activities of molecular networks that execute metabolic or cytoskeletal processes, or regulate these by signal transduction, are altered in a complex manner by diverse genetic mutations in concert with the environmental context. A major challenge therefore is how to develop actionable understanding of this multi-variate dysregulation, with respect both to how it arises from diverse genetic mutations and to how it may be ameliorated by prospective treatments. While high-throughput experimental platform technologies ranging from genomic sequencing to transcriptomic, proteomic, and metabolomic profiling are now commonly used for molecular-level characterization of tumor cells and surrounding tissues, the resulting data sets defy straight-forward intuitive interpretation with respect to potential therapeutic targets or the effects of perturbation. In this review article we will discuss how significant advances can be obtained by applying computational modeling approaches to elucidate the pathways most critically involved in tumor formation and progression, impact of particular mutations on pathway operation, consequences of altered cell behavior in tissue environments, and effects of molecular therapeutics.

Rosiglitazone Prevents the Progression of Pre-invasive Lung Cancer in a Murine Model

Tue, 27 Oct 2009 12:30:55 PDT

There is a critical need to identify efficacious chemopreventive agents for lung cancer that can be taken chronically with no side effects and whose mechanisms of action do not involve genotoxicity that could drive, rather than impede cancer progression. We evaluated the ability of a chemopreventive cocktail that included selenium (antioxidant), rosiglitazone (PPAR agonist), sodium phenylbutyrate or valproic acid (histone deacetylase inhibitors) and hydralazine (cytosine demethylating agent) to prevent the progression of lung cancer in A/J mice treated with NNK. Agents were administered alone or in various combinations. Effects of the chemopreventive agents were quantified based on the proportion of hyperplasias and adenomas within the mouse lung. Significant effects on tumor progression were seen in all treatment groups that included rosiglitazone as reflected by a 47–57% increase in number of hyperplasias and a 10–30% decrease in adenomas. Cell proliferation was also reduced in these treatment groups by approximately 40%. Interestingly, while treatment with rosiglitazone alone did not significantly affect lesion size, striking effects were seen in the combination therapy group that included sodium phenylbutyrate, with the volume of hyperplasias and adenomas decreasing by 40% and 77%, respectively. These studies demonstrate for the first time that chronic in vivo administration of rosiglitazone, used in the management of diabetis mellitus, can significantly block the progression of premalignant lung cancer in the A/J mouse model.

Increasing fish consumption does not affect genotoxicity markers in the colon in an intervention study

Tue, 27 Oct 2009 12:30:52 PDT

Observational studies suggest that fish consumption is associated with a decreased colorectal cancer (CRC) risk. A possible mechanism by which fish could reduce CRC risk is by decreasing colonic genotoxicity. However, concerns have also been raised over the levels of toxic compounds found in mainly oil-rich fish, which could increase genotoxicity. Therefore, the objective was to investigate the effects of fish on genotoxicity markers in the colon in a randomized controlled parallel intervention study. For a period of six months, subjects were randomly allocated to receive two extra weekly portions of (i) oil-rich fish (salmon), (ii) lean fish (cod), or (iii) just dietary advice. The Comet Assay was used to measure the DNA damage-inducing potential of fecal water (n=89) and DNA damage in colonocytes (n=70) collected pre- and post-intervention as markers of genotoxicity.

Genotoxicity of fecal water was not markedly changed after fish consumption: 1.0% increase in tail intensity (TI) (95% confidence interval (CI) -5.1; 7.0) in the salmon group and 0.4% increase in TI (95% CI -5.3; 6.1) in the cod group compared with the dietary advice group. DNA damage in colonocytes was also not significantly changed after fish consumption, in either the salmon group, (-0.5%TI, 95% CI -6.9; 6.0), or cod group (-3.3%TI, 95% CI -10.8; 4.3) compared with the dietary advice group. Measurements of genotoxicity of fecal water and DNA damage in colonocytes did not correlate (r=0.06, n=34). In conclusion, increasing consumption of either oil-rich or lean fish did not affect genotoxicity markers in the colon.

Cancer Stem Cells : A Reality, A Myth, A Fuzzy Concept or A Misnomer ? An Analysis

Maenhaut, C., Dumont, J.E., Roger, P., van Staveren, W.C.G. — Sun, 25 Oct 2009 19:48:03 PDT

The concept of cancer stem cells (CSC) embodies two aspects: the stem cell as the initial target of the oncogenic process and the existence of two populations of cells in cancers: the CSC and derived cells. The second is discussed in this review.

CSC are defined as cells having three properties: a selectively endowed tumorigenic capacity, an ability to recreate the full repertoire of cancer cells of the parent tumor, and the expression of a distinctive repertoire of surface biomarkers. In operational terms the CSC are among all cancer cells those able to initiate a xenotransplant. Other explicit or implicit assumptions exist, including the concept of CSC as a single unique infrequent population of cells. To avoid such assumptions we propose to use the operational term tumor propagating cells (TPC); indeed the cells that initiate transplants did not initiate the cancer.

The experimental evidence supporting the explicit definition is analyzed. Cancers indeed contain a fraction of cells mainly responsible for the tumor development. However there is evidence that these cells do not represent one homogeneous population. Moreover there is no evidence that the derived cells result from an asymmetric, qualitative and irreversible process. A more general model is proposed of which the CSC model could be one extreme case. We propose that the TPC are multiple evolutionary selected cancer cells with the most competitive properties, (maintained by (epi-)genetic mechanisms), at least partially reversible, quantitative rather than qualitative and resulting from a stochastic rather than deterministic process.

Common Genetic Variation in IGF1, IGFBP1, and IGFBP3 and Ovarian Cancer Risk

Terry, K. L., Tworoger, S. S., Gates, M. A., Cramer, D. W., Hankinson, S. E. — Sun, 25 Oct 2009 19:48:01 PDT

IGF1 and its binding proteins foster cellular proliferation and inhibit apoptosis. In vitro studies show that IGF1 increases ovarian cell growth and invasive potential, suggesting a role for the IGF1 pathway in ovarian cancer etiology. We evaluated genetic variation in the IGF1, IGFBP1, and IGFBP3 genes in relation to ovarian cancer risk by genotyping 29 haplotype-tagging SNPs in 1173 cases and 1201 controls from the New England Case Control Study (NECC) and 296 cases and 854 controls from the Nurses’ Health Study (NHS). The association of haplotypes and SNPs with ovarian cancer was estimated using unconditional (NECC) and conditional (NHS) logistic regression. Additionally, we evaluated the association of SNPs with IGF1, IGFBP3, and IGFBP2 plasma levels (n = 380 NHS controls). Our data suggest a decreased risk for women carrying haplotype 2C of the IGF1 gene (OR = 0.82, 95% CI = 0.69-0.98), and an increased risk for women carrying haplotype 1D (OR = 1.41, 95% CI = 1.03-1.94) or 2D (OR = 1.20, 95% CI = 1.01-1.41) in the binding proteins. When evaluated individually, three SNPs in the IGF binding proteins (rs10228265, rs4988515, rs227062) were associated with increased ovarian cancer risk, and several IGF1 (rs11111285, rs1996656, rs1019731) and IGFBP3 (rs2270628, rs2854746, rs2854744) SNPs were significantly associated with IGF1, IGFBP3, and IGFBP2 plasma levels. Some haplotypes and SNPs in the IGF pathway genes may be associated with ovarian cancer risk; however, these results need to be confirmed. Of particular interest was the IGFBP3 SNP rs2270628 which was associated with both increased IGF1 plasma levels and higher ovarian cancer risk.

Basic properties and molecular mechanisms of exogenous chemical carcinogens

Irigaray, P., Belpomme, D. — Sun, 25 Oct 2009 19:47:59 PDT

Exogenous chemical carcinogenesis is an extremely complex multifactorial process during which gene-environment interactions involving chronic exposure to exogenous chemical carcinogens (ECCs) and polymorphisms of cancer susceptibility genes add further complexity. We describe the properties and molecular mechanisms of ECCs that contribute to induce and generate cancer. A basic and specific property of many lipophilic organic ECCs including polycyclic aromatic hydrocarbons and polyhalogenated aromatic hydrocarbons is their ability to bioaccumulate in the adipose tissue, from where they may be released in the blood circulation and target peripheral tissues for carcinogenesis. Many organic ECCs are procarcinogens and consequently need to be activated by the cytochrome P450 (CYP) system and/or other enzymes before they can adduct DNA and proteins. Because they contribute not only to the cocarcinogenic and promoting effects of many aromatic pollutants, but also to their mutagenic effects, the aryl hydrocarbon receptor (AhR)-activating and the inducible CYP systems are central to exogenous chemical carcinogenesis. Another basic property of ECCs is their ability to induce stable and bulky DNA adducts which cannot be simply repaired by the different repair systems. In addition, following ECC exposure mutagenesis may also be caused indirectly by free radical production and by epigenetic alterations. As a result of complex molecular interplays, direct and/or indirect mutagenesis may especially account for the carcinogenic effects of many exogenous metals and metalloids. Because of these molecular properties and action mechanisms, we conclude that ECCs could be major contributors to human cancer, with obviously great public health consequences.

Loss of imprinting of the insulin-like growth factor II (IGF2) gene in esophageal normal and adenocarcinoma tissues

Zhao, R., DeCoteau, J., Geyer, C. R., Gao, M., Cui, H., Casson, A. G. — Tue, 20 Oct 2009 08:33:11 PDT

To evaluate loss of imprinting and expression of the IGF2 gene in matched esophageal normal and adenocarcinoma tissues, we studied a prospective cohort of 77 patients who underwent esophageal resection between 1998 and 2003. IGF2 imprinting status was determined by RT-PCR following Apa I digestion, and quantitative polymerase chain reaction was used to evaluate IGF2 expression, which was correlated with clinico-pathologic findings, disease-free and overall survival. 32% (14/44) of informative tissues showed loss of IGF2 imprinting, with a strong correlation between the tumor and normal esophageal epithelia (Kappa = 0.89, p<0.01). Normal epithelia with loss of imprinting had increased expression of IGF2 (median: 2.91, 95%CI: 0.93-5.06) compared with imprinted normal epithelia (median: 1.13, 95%CI: 0.85-1.39) (p = 0.03). In contrast, tumors with loss of imprinting had significantly reduced IGF2 expression (median: 1.87, 95%CI: 0.53-5.21) compared with normally imprinted tumors (median: 6.79, 95%CI: 3.39-15.89) (p = 0.016). Patients below the age of 65 years with normally imprinted tumors had significantly reduced 5-year disease free survival (24%) compared with patients whose tumors had loss of imprinting for IGF2 (55%) (p = 0.03). Cox regression analysis showed IGF2 overexpression was associated with significantly reduced disease free survival (p = 0.04). We conclude that in a subgroup of younger patients, loss of IGF2 imprinting was associated with improved outcome following esophageal resection. Expression of IGF2 in esophageal adenocarcinoma and normal esophageal epithelia depended on imprinting status and tissue type, suggesting novel molecular regulatory mechanisms in esophageal tumorigenesis.

Cucurbitacin I Elicits Anoikis-sensitization, Inhibits Cellular Invasion and In Vivo Tumor Formation Ability of Nasopharyngeal Carcinoma Cells

Tue, 20 Oct 2009 08:33:10 PDT

Nasopharyngeal carcinoma (NPC) is an Asian-prevalent head and neck cancer with high invasiveness. Although several important risk factors for NPC development have been identified, there is currently no preventive strategy for NPC, even in endemic regions. Signal Transducer and Activator of Transcription 3 (STAT3) has been implicated in NPC carcinogenesis, which may serve as a potential target for cancer prevention. Here, we examined the chemopreventive potential of Cucurbitacin I, a natural-occurring selective inhibitor of JAK/STAT3, in NPC models. We hypothesized that Cucurbitacin I would prevent NPC invasion and tumor-formation. Our data demonstrated that brief exposure of NPC cells to Cucurbitacin I was sufficient to significantly reduce the in vitro clonogenicity and in vivo tumorigenicity of NPC cells. The chemopreventive potential of Cucurbitacin I was further demonstrated by pre-dosing of the animal with Cucurbitacin I prior to tumor inoculation, which was found to be able to suppress tumor growth up to 7 days post-inoculation. The anti-proliferation activity of Cucurbitacin I was accompanied by downregulation of phospho-STAT3, and STAT3 target genes (e.g. cyclin D1 and Mcl-1). Cucurbitacin I also reduced the invasiveness of invasive NPC cell lines with elevated STAT3 activation. Furthermore, our data demonstrated for the first time that Cucurbitacin I harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced cell death) against human cancer. Taken together, our results suggested that Cucurbitacin I may be a potent chemopreventive agent for NPC with anti-invasion and anoikis-sensitizing activities.

Abl Interactor 1 Regulates Src-Id1-Matrix Metalloproteinase 9 Axis and Is Required for Invadopodia Formation, Extracellular Matrix Degradation, and Tumor Growth of Human Breast Cancer Cells

Sun, X., Li, C., Zhuang, C., Gilmore, W. C., Cobos, E., Tao, Y., Dai, Z. — Tue, 20 Oct 2009 08:33:10 PDT

Abl interactor 1 (Abi1) is a key regulator of actin polymerization/depolymerization. The involvement of Abi1 in the development of abnormal cytoskeletal functions of cancer cells has recently been reported. It remains unclear, however, how Abi1 exerts its effects in tumor cells and whether it contributes to tumor progression in vivo. We report here a novel function for Abi1 in the regulation of invadopodia formation and Src-Id1-matrix metalloproteinase 9 (MMP-9) pathway in MDA-MB231 human breast cancer cells. Abi1 is found in the invadopodia of MDA-MB231 cells. Epigenetic silencing of the Abi1 gene by short hairpin RNA (shRNA) in MDA-MB231 cells impaired the formation of invadopodia and resulted in down-regulation of the Scr activation and Id1/MMP-9 expression. The decreased invadopodia formation and MMP-9 expression correlate with a reduction in the ability of these cells to degrade extracellular matrix (ECM). Remarkably, the knockdown of Abi1 expression inhibited tumor cell proliferation and migration in vitro, and slowed tumor growth in vivo. Taken together, these results indicate that the Abi1 signaling plays a critical role in breast cancer progression and suggest that this pathway may serve as a therapeutic target for the treatment of human breast cancer.

miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma

Furuta, M., Kozaki, K.-i., Tanaka, S., Arii, S., Imoto, I., Inazawa, J. — Tue, 20 Oct 2009 08:33:09 PDT

MicroRNAs (miRNAs) are a class of small non-coding RNAs that, in general, negatively regulate gene expression. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. Some miRNA genes harboring CpG-islands undergo methylation-mediated silencing, a characteristic of many tumor-suppressor genes. To identify such miRNAs in hepatocellular carcinoma (HCC), we first examined the methylation status of 43 loci containing CpG-islands around 39 mature miRNA genes in a panel of HCC cell lines and non-cancerous liver tissues as controls. Among 11 miRNA genes frequently methylated in HCC cell lines but not in non-cancerous liver tissues, 3 miRNA genes, i.e. miR-124, miR-203, and miR-375, were selected as silenced miRNAs through CpG-island methylation by comparing methylation and expression status and evaluating restored expression after treatment with 5-aza-2'-deoxycytidine. In primary tumors of HCC with paired non-tumorous liver tissues, only miR-124 and miR-203 showed frequent tumor-specific methylation, and their expression status was inversely correlated with methylation status. Ectopic expression of miR-124 or miR-203 in HCC cells lacking their expression inhibited cell growth, with direct down-regulation of possible targets, cyclin-dependent kinase 6 (CDK6), vimentin (VIM), SET and MYND domain containing 3 (SMYD3), and IQ motif containing GTPase activating protein 1 (IQGAP1) or ATP-binding cassette, sub-family E, member 1 (ABCE1), respectively. Our results suggest that miR-124 and miR-203 are novel tumor-suppressive miRNAs for HCC epigenetically silenced and activating multiple targets during hepatocarcinogenesis.

A novel oncolytic adenovirus selectively silences the expression of tumor-associated STAT3 and exhibits potent anti-tumoral activity

Tue, 20 Oct 2009 08:33:08 PDT

Tumor cells acquire the ability to proliferate uncontrollably, resist apoptosis, sustain angiogenesis and evade immune surveillance. STAT3 regulates all of these processes in a surprisingly large number of human cancers. Consequently, the STAT3 protein is emerging as an ideal target for cancer therapy. This paper reports the generation of an oncolytic adenovirus (M4), which selectively blocks STAT3 signaling in tumor cells as a novel therapeutic strategy. M4 selectively replicated in tumor cells and expressed high levels of antisense STAT3 cDNA during the late phase of the viral infection in a replication-dependent manner. The viral progeny yield of M4 in tumor cells was much higher than that of the parent adenoviral mutants, Ad5/dE1A. M4 effectively silenced STAT3 and its target genes in tumor cells while sparing normal cells and exhibited potent anti-tumoral efficacy in vitro and in vivo. Systemic administration of M4 significantly inhibited tumor growth in an orthotopic gastric carcinoma mouse model, eliminated abdominal cavity metastases and prolonged survival time. In summary, M4 has low toxicity and great potential as a therapeutic agent for different types of cancers.

Lung cancer susceptibility among atomic-bomb survivors in relation to CA repeat number polymorphism of epidermal growth factor receptor gene and radiation dose

Tue, 20 Oct 2009 08:33:08 PDT

Lung cancer is a leading cause of cancer death worldwide. Prevention could be improved by identifying susceptible individuals as well as improving understanding of interactions between genes and etiologic environmental agents, including radiation exposure. The EGFR signaling pathway, regulating cellular radiation sensitivity, is an oncogenic cascade involved in lung cancer, especially adenocarcinoma. The CA repeat number polymorphism in the first intron of EGFR has been shown to be inversely correlated with EGFR production. It is hypothesized that CA repeat number may modulate individual susceptibility to lung cancer. Thus, we carried out a case-cohort study within the Japanese atomic-bomb survivor cohort to evaluate a possible association of CA repeat polymorphism with lung cancer risk in radiation-exposed or negligibly exposed (< 5 mGy) atomic-bomb survivors. First, by dividing study subjects into Short and Long genotypes–defined as the summed CA repeat number of two alleles ≤ 37 and ≥ 38, respectively–we found that the Short genotype was significantly associated with an increased risk of lung cancer, specifically adenocarcinoma, among negligibly-exposed subjects. Next we found that prior radiation exposure significantly enhanced lung cancer risk of survivors with the Long genotype, while the risk for the Short genotype did not show any significant increase with radiation dose, resulting in indistinguishable risks between these genotypes at a high radiation dose. Our findings imply that the EGFR pathway plays a crucial role in assessing individual susceptibility to lung adenocarcinoma in relation to radiation exposure.

n-3 polyunsaturated fatty acids modulate carcinogen-directed non-coding microRNA signatures in rat colon

Tue, 13 Oct 2009 06:36:36 PDT

We have hypothesized that dietary modulation of intestinal non-coding RNA (microRNA) expression may contribute to the chemo-protective effects of nutritional bioactives (fish oil, pectin). To fully understand the effects of these agents on the expression of microRNAs, Sprague Dawley rats were fed diets containing corn oil or fish oil with pectin or cellulose and injected with azoxymethane (AOM, a colon-specific carcinogen) or saline (control). Real time PCR using microRNA specific primers and Taq Man^TM probes was carried out to quantify effects on microRNA expression in colonic mucosa. From 368 mature microRNAs assayed, at an early stage of cancer progression (10 wk post AOM injection), let-7d, miR-15b, miR-107, miR-191 and miR-324-5p were significantly (p<0.05) affected by diet x carcinogen interactions. Overall, fish oil fed animals exhibited the smallest number of differentially expressed microRNAs (AOM vs saline treatment). With respect to the tumor stage (34 wk post AOM injection), 46 microRNAs were dysregulated in adenocarcinomas compared to normal mucosa from saline-injected animals. Of the 27 microRNAs expressed at higher (p<0.05) levels in tumors, miR-34a, 132, 223, and 224 were over expressed at greater than 10-fold. In contrast, the expression levels of miR-192, 194, 215, and 375 were dramatically reduced (0.32-fold or lower) in adenocarcinomas. These results demonstrate for the first time the utility of the rat AOM model, and the novel role of fish oil in protecting the colon from carcinogen-induced microRNA dysregulation.

Vascular endothelial growth factor-c (VEGF-C) promotes angiogenesis by induction of COX-2 in leukemic cells via the VEGF-R3/JNK/AP-1 pathway

Tue, 13 Oct 2009 06:36:35 PDT

Vascular endothelial growth factor (VEGF)-C is recognized as a tumor lymphangiogenic factor based on the effects of activated VEGF-R3 on lymphatic endothelial cells. Many tumor cells express VEGF-R3, but the function of this receptor in tumor cells is largely unknown. It has been reported that the VEGF-C/VEGF-R3 axis is activated in subsets of leukemia patients. Herein we have shown that VEGF-C induces angiogenic activity in the tube formation assay in vitro and Matrigel plug assay in vivo by up-regulating an angiogenic factor, cyclooxygenase-2 (COX-2), through VEGF-R3 in the human acute myeloid leukemia (AML) cell line, THP-1. COX-2 induction by VEGF-C was also observed in other VEGF-R3⁺ human AML cell lines (U937 and HL60). Moreover, immunohistochemical analysis of bone marrow specimens of 37 patients diagnosed with AML revealed that VEGF-C expression in specimens was associated with the expression of COX-2 (P < 0.001). The manner by which signaling pathways transduced by VEGF-C is responsible for COX-2 up-regulation was further investigated. Blocking the p42/44 MAPK pathway with the MEK inhibitor, PD98059, failed to inhibit VEGF-C-mediated COX-2 expression. However, VEGF-C-induced COX-2 up-regulation was effectively abolished by overexpression of dominant-negative (dn) JNK or treatment with the JNK inhibitor, SP600125. VEGF-C induced JNK-dependent nuclear translocation of c-Jun. Furthermore, chromatin immunoprecipitation and reporter assays revealed that VEGF-C enhanced c-Jun binding to the cyclic AMP–response element (CRE) of the COX-2 promoter and induced COX-2 expression. In sum, the data herein highlight the pathogenic role of VEGF-C in leukemia via regulation of angiogenesis through up-regulation of COX-2.

Dietary olive oil and corn oil differentially affect experimental breast cancer through distinct modulation of the p21ras signaling and the proliferation-apoptosis balance

Solanas, M., Grau, L., Moral, R., Vela, E., Escrich, R., Escrich., E. — Tue, 13 Oct 2009 06:36:34 PDT

Extra-virgin olive oil (EVOO) has been hypothesized to have chemopreventive effects on breast cancer, unlike high corn oil (HCO) diets which stimulate it. We have investigated mechanisms of these differential modulatory actions on experimental mammary cancer. In dimethylbenz()anthracene-induced breast adenocarcinomas of rats fed a high EVOO, HCO and control diets (n = 20 for each group), we have analyzed the expression and activity of ErbB receptors, p21Ras and its ERK1/2, Akt and RalA/B effectors by immunoblotting analyses. We explored the Ha-ras1 mutation status by Southern blot, MAMA and sequencing, and the HMG-CoA reductase and squalene synthase mRNA expression by Real Time-polymerase chain reaction (PCR). We analyzed the tumor mitotic index, PCNA levels, and apoptosis through Caspase-3 analysis and TUNEL assays. Finally, we measured the 8-oxo-2’-deoxyguanosine levels. Non-parametrical statistics were used. The EVOO diet decreased Ras activation, down-regulated the Ras/PI3K/Akt pathway and up-regulated the Raf/Erk pathway, compared to the control. In contrast, the HCO diet did not modify Ras activity but rather enhanced the Raf/Erk pathway. The EVOO diet decreased the cleaved ErbB4 levels, compared to the HCO diet, increased apoptosis and diminished the mono-ubiquitylated PCNA levels, which is related to DNA damage. Tumors from rats fed the EVOO diet displayed a more benign phenotype, whereas those from rats fed the HCO diet were biologically more aggressive. In conclusion, high EVOO and corn oil diets exert their modulatory effects on breast cancer through a different combination of Ras signaling pathways, a different proliferation/apoptosis balance and probably distinct levels of DNA damage.

MK2 regulates the early stages of skin tumor promotion

Tue, 06 Oct 2009 07:00:54 PDT

The association between inflammation and tumorigenesis is well recognized. Mitogen-activated protein kinase-activated protein kinase-2 (MK2) is known to play a pivotal role in inflammatory processes. Here we studied the effect of MK2-deficiency and TNF--deficiency on skin tumor development in mice using the two-stage chemical carcinogenesis model. We found that MK2^-/- mice developed significantly fewer skin tumors compared with both TNF-^-/- and wild-type mice when induced by initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and by promotion with 12-O-tetradecanoylphorbol-13-acetat (TPA). The TPA-induced inflammatory response was reduced in both, TNF-^-/- mice and MK2^-/- mice, but most pronounced in TNF-^-/- mice, indicating that a reduced inflammatory response was not the only explanation for the inhibited tumorigenesis seen in MK2^-/- mice. Interestingly, increased numbers of apoptotic cells were detected in the epidermis of MK2^-/- mice compared with TNF-^-/- and wild-type mice, suggesting an additional role of MK2 in the regulation of apoptosis. This was further supported by a): increased levels of the tumor suppressor protein p53 in MK2^-/- mice after DMBA/TPA treatment compared with controls, b): reduced phosphorylation (activation) of the negative p53 regulator, murine double minute 2 (Mdm2) in MK2^-/- mouse keratinocytes in vitro and c): a significant decrease in the DMBA/TPA induced apoptosis in cultured MK2^-/- keratinocytes transfected with p53 siRNA. Taken together, these findings demonstrate a dual role of MK2 in the early stages of tumor promotion through regulation of both the inflammatory response and apoptosis of DNA-damaged cells. These results also identify MK2 as a putative target for future skin carcinoma therapy.

A Case-Control and a Family-Based Association Study Revealing an Association between CYP2E1 Polymorphisms and Nasopharyngeal Carcinoma Risk in Cantonese

Mon, 05 Oct 2009 09:59:24 PDT

Nasopharyngeal carcinoma (NPC) is rare in most parts of the world but is more prevalent in Southern China, especially in Guangdong. The cytochrome P450 2E1 (CYP2E1) has been recognized as one of the critically important enzymes involved in oxidizing carcinogens, and is likely to be associated with NPC carcinogenesis. To systematically investigate the association between genetic variants in CYP2E1 and NPC risk in Cantonese, two independent studies, a family-based association study and a case-control study, were conducted using the haplotype-tagging SNP approach. A total of 2,499 individuals from 546 nuclear families were initially genotyped for the family-based association study. SNPs rs9418990, rs915906, rs915908, rs8192780, rs1536826, rs3827688 and one haplotype h2 (CGTGTTAA) were revealed to be significantly associated with the NPC phenotype (P = 0.045 to 0.003 and P = 0.003, respectively). To follow up the initial study, a case-control study including 755 cases and 755 controls was conducted. Similar results were observed in the case-control study in individuals younger than 46 years of age and had a history of cigarette smoking, with ORs of specific genotypes ranging from 1.88 to 2.99 corresponding to SNP rs9418990, rs3813865, rs915906, rs2249695, rs8192780, rs1536826, rs3827688, and of haplotypes h2 with OR = 1.65 (P = 0.026), h5 (CCCGTTAA) with OR = 2.58 (P = 0.007). The values of false-positive report probability were less than 0.015 for six SNPs, suggesting that the reported associations are less likely to be false. This study provides robust evidence for associations between genetic variants of CYP2E1 and NPC risk.

ELR+ CXC chemokines and oncogenic Ras-mediated tumorigenesis

O'Hayer, K., Brady, D. C., Counter, C. M. — Mon, 05 Oct 2009 09:59:23 PDT

The small GTPase Ras is mutated to remain in the active oncogenic state in one third of human cancers, thereby promoting tumorigenesis. It has recently come to light that one consequence of oncogenic Ras signaling is secretion of cytokines VEGF, IL6, hCXCL1, and hCXCL8. As the latter two belong to the ELR+ CXC chemokine family, we investigated whether the entire family of ELR+ CXC chemokines plays a role in oncogenic Ras-mediated tumorigenesis. We now demonstrate that oncogenic Ras induced the expression and secretion of the ELR+ CXC chemokine family in different tumorigenic human cells, and that these chemokines are elevated in tumor specimens. Moreover, genetic ablation of the common receptor for these chemokines, mCXCR2, reduced oncogenic Ras-driven tumorigenesis in mice. Taken together, we suggest that oncogenic Ras induces the secretion of the ELR+ CXC chemokine family to promote tumorigenesis. This chemokine signature may identify the presence of Ras activation in cancer and perhaps even serve as targets for oncogenic Ras-driven tumor cells.

IkappaBalpha gene promoter polymorphisms are associated with hepatocarcinogenesis in patients infected with hepatitis B virus genotype C

Thu, 01 Oct 2009 06:23:08 PDT

Genetic predisposition of nuclear factor kappaB signaling pathways linking inflammation to hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) remains unresolved. We conducted a case-control study to determine the associations of the polymorphisms within the promoter regions of NFKB1 encoding nuclear factor kappaB1 and NFKBIA encoding IkappaBalpha with the development of HCC. A total of 404 healthy controls, 482 non-HCC subjects with HBV infection, and 202 patients with HCC were included. NFKB1-94ATTG2 allele and GG allele in the 3' untranslated region of NFKBIA were more prevalent in HCC patients than in the healthy controls. NFKBIA-826CT and NFKBIA-881AG allelic carriages were more prevalent in HCC patients than in the non-HCC subjects with HBV infection. The estimated haplotype frequency of NFKBIA promoter -881G-826T-519C was significantly higher in the patients with HCC than in the HBV-infected subjects without HCC (odds ratio = 3.142, P = 0.002). As compared with the HBV-infected subjects without HCC, NFKBIA -826 T and NFKBIA -881 AG allelic carriages were only associated with HCC risk in the subjects with HBV genotype C. The association of NFKBIA -881AG allelic carriage with HCC risk was not affected by liver cirrhosis status, alanine aminotransferase level, and hepatitis B e antigen status. By multivariate regression analysis, NFKB1-94ATTG2, NFKBIA-826T, NFKBIA-881AG, and HBV genotype C were independently associated with an increased risk of HCC. In conclusion, NFKB1-94ATTG2 allele and haplotype -881G-826T-519C in NFKBIA promoter were associated with hepatocarcinogenesis. NFKBIA-826T and -881AG were associated with the risk of HCC in the subjects infected with HBV genotype C.

Role of Notch signaling in colorectal cancer

Qiao, L., Wong, B. C. — Wed, 30 Sep 2009 05:21:34 PDT

Nrf2: Friend or Foe for Chemoprevention?

Kensler, T. W., Wakabayashi, N. — Wed, 30 Sep 2009 05:21:33 PDT

Health reflects the ability of an organism to adapt to stress. Stresses – metabolic, proteotoxic, mitotic, oxidative and DNA-damage stresses – not only contribute to the etiology of cancer and other chronic degenerative diseases, but are also hallmarks of the cancer phenotype. Activation of the KEAP1-NRF2 signaling pathway is an adaptive response to environmental and endogenous stresses, and serves to render animals resistant to chemical carcinogenesis and other forms of toxicity, whilst disruption of the pathway exacerbates these outcomes. This pathway can be induced by thiol-reactive small molecules that demonstrate protective efficacy in preclinical chemoprevention models and in clinical trials. However, mutations and epigenetic modifications affecting the regulation and fate of NRF2 can lead to constitutive dominant hyper-activation of signaling that preserves rather than attenuates cancer phenotypes by providing selective resistance to stresses. This review provides a synopsis of KEAP1-NRF2 signaling, compares the impact of genetic versus pharmacologic activation, and considers both the attributes and concerns of targeting the pathway in chemoprevention.

Curcumin Inhibits COPD-Like Airway Inflammation and Lung Cancer Progression in Mice

Wed, 30 Sep 2009 05:21:33 PDT

Recent studies have demonstrated that K-ras mutations in lung epithelial cells elicit inflammation that promotes carcinogenesis in mice (intrinsic inflammation). The finding that patients with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer after controlling for smoking suggests a further link between lung cancer and extrinsic inflammation. Besides exposure to cigarette smoke, it is thought that airway inflammation in COPD is caused by bacterial colonization, particularly with non-typeable Hemophilus influenzae (NTHi). Previously, we have shown that NTHi-induced COPD-like airway inflammation promotes lung cancer in an airway conditional K-ras-induced mouse model. To further test the role of inflammation in cancer promotion, we administered the natural anti-inflammatory agent, curcumin, 1% in diet before and during weekly NTHi exposure. This significantly reduced the number of visible lung tumors in the absence of NTHi exposure by 85% and in the presence of NTHi exposures by 53%. Mechanistically, curcumin markedly suppressed NTHi-induced increased levels of the neutrophil chemoattractant KC by 80% and neutrophils by 87% in bronchoalveolar lavage fluid. In vitro studies of murine K-ras-induced lung adenocarcinoma cell lines (LKR10 and LKR13) indicated direct anti-tumoral effects of curcumin by reducing cell viability, colony formation and inducing apoptosis. We conclude that curcumin suppresses the progression of K-ras induced lung cancer in mice by inhibiting intrinsic and extrinsic inflammation and by direct anti-tumoral effects. These findings suggest that curcumin could be used to protract the premalignant phase and inhibit lung cancer progression in high-risk COPD patients.

The extracellular loop 2 of TM4SF5 inhibits integrin {alpha}2 on hepatocytes under collagen type I environment

Tue, 29 Sep 2009 07:30:08 PDT

Four-transmembrane L6 family member 5 (TM4SF5) and its homologue L6, a tumor antigen, form a four-transmembrane L6 family. TM4SF5 expression causes uncontrolled cell proliferation and angiogenesis. Although other genuine transmembrane 4 superfamily (TM4SF) members cooperate with integrins for cell migration, roles of TM4SF5 in the cellular spreading and migration are unknown. Using hepatocarcinoma cell clones that ectopically express TM4SF5, we found that cross-talks via an extracellular interaction between TM4SF5 and integrin 2 in collagen type I-environment inhibited integrin 2 functions such as spreading on and migration toward collagen I, which were recovered by suppression of TM4SF5 or structural disturbance of its second extracellular loop using a peptide or mutagenesis. Altogether, the observations suggest that TM4SF5 in hepatocytes negatively regulates integrin 2 function via an interaction between the extracellular loop 2 of TM4SF5 and integrin 2 during cell spreading on and migration through collagen I environment.

HIF-1 is induced via EGFR activation and mediates resistance to anoikis-like cell death under lipid rafts/caveolae-disrupting stress

Tue, 29 Sep 2009 07:30:07 PDT

The plasma membrane microdomains, lipid rafts, are involved in regulation of cellular functions such as cell survival and adhesion. Cholesterol is a critical component of lipid rafts in terms of their integrity and functions and rafts disruption by cholesterol depletion can induce detachment-induced cell death. Hypoxia inducible factor-1 (HIF-1) is stabilized in hypoxia and transactivates numerous genes required for cellular adaptation to hypoxia. It is also induced by non-hypoxic stimuli and contributes to cell survival. Because hypoxia inhibits cholesterol synthesis and HIF-1 plays a role in this process, we here explored a possible connection between lipid rafts and HIF-1. We investigated whether HIF-1 is regulated during cholesterol depletion/rafts disruption in A431 cells in normoxic conditions. Methyl-beta cyclodextrin (MβCD), which induces cholesterol depletion, up-regulated HIF-1 even under normoxic conditions and this up-regulation required EGF receptor and ERK1&2 activation, but not Akt activation. MβCD treatment induced HIF-1 up-regulation at both the transcriptional and translational levels, but not at the post-translational levels. In addition, MβCD robustly induced VEGF production and stimulated a HRE-driven luciferase reporter activity under normoxic conditions, indicating that MβCD-induced HIF-1 is functionally activated. Both EGF receptor activity and HIF-1 expression were higher in the attached cells than in the detached cells after MβCD treatment. Furthermore, inhibition of HIF-1 by RNA interference accelerated cell detachment, thus increasing cell death, indicating that HIF-1 expression attenuates MβCD-induced anoikis-like cell death. These data suggest that, depending on cholesterol levels, lipid rafts or membrane fluidity are likely to regulate HIF-1 expression in normoxia by modulating rafts protein activities such as EGF receptor, and this connection between lipid rafts and HIF-1 regulation may provide cell survival under membrane-disturbing stress.

The Rac1/MKK7/JNK pathway signals upregulation of Atg5 and subsequent autophagic cell death in response to oncogenic Ras

Sat, 26 Sep 2009 04:10:36 PDT

To prevent the development of malignancies, mammalian cells activate disposal programs, such as programmed cell death, in response to deregulated oncogene expression. However, the molecular basis for regulation of cellular disposal machinery in response to activated oncogenes is unclear at present. In this study, we show that upregulation of the autophagy-related protein, Atg5, is critically required for the oncogenic H-ras-induced autophagic cell death, and that Rac1/MKK7/JNK signals upregulation of Atg5. Overexpression of H-ras^V12 induced marked autophagic vacuole formation and cell death in normal fibroblasts, which remained unaffected by a caspase inhibitor. Pretreatment with Bafilomycin A1, an autophagy inhibitor, completely attenuated H-ras^V12-induced cell death as well as autophagic vacuole formation. Selective production of Atg5 was observed in cells overexpressing H-ras^V12, and siRNA targeting of Atg5 clearly inhibited autophagic cell death. Interestingly, inhibition of JNK or c-Jun by specific siRNA suppressed Atg5 upregulation and autophagic cell death. Moreover, inhibition of MKK7, but not MKK4, effectively attenuated H-ras^V12-induced JNK activation. In addition, ectopic expression of RacN17 or Rac1-siRNA effectively inhibited MKK7-JNK activation, Atg5 upregulation, and autophagic cell death. These data support the notion that upregulation of Atg5 is required for the oncogenic H-ras-induced autophagic cell death in normal fibroblasts, and that activation of Rac1/MKK7/JNK signaling pathway leads to upregulation of Atg5 in response to oncogenic H-ras. Our findings suggest that in cells acquiring deregulated oncogene expression, oncogenic stress triggers autophagic cell death, which protects cells against malignant progression.

Delphinidin suppresses ultraviolet B-induced cyclooxygenases-2 expression through inhibition of MKK4 and PI-3 kinase

Wed, 23 Sep 2009 07:34:44 PDT

Cyclooxygenase-2 (COX-2), a key mediator of inflammation, and its product, prostaglandin E₂ (PGE₂), enhance carcinogenesis, particularly in skin. Ultraviolet (UV)B is the most carcinogenic component of solar irradiation, and a crucial role of COX-2 in UVB-mediated skin carcinogenesis has been reported. Here we investigated the effects of delphinidin, an abundant dietary anthocyanin, on UVB-induced COX-2 up-regulation and the underlying molecular mechanism. We found that delphinidin suppressed UVB-induced COX-2 expression in JB6 P+ mouse epidermal cells. COX-2 promoter activity and PGE₂ production were also suppressed by delphinidin treatment within noncytotoxic concentrations. Activator protein-1 and nuclear factor-B, crucial transcription factors involved in COX-2 expression, were activated by UVB, and delphinidin abolished this activation. UVB-induced phosphorylation of c-Jun N-terminal kinase, p38 kinase, and Akt was inhibited by delphinidin. The activities of mitogen-activated protein kinase kinase 4 (MKK4) and phosphatidylinositol-3 (PI-3) kinase were inhibited markedly by delphinidin. A pull-down assay using delphinidin-Sepharose beads revealed that delphinidin binds directly with MKK4 or PI-3 kinase in a manner that was competitive with ATP. Moreover, in vivo investigations using mouse skin revealed that the upregulation of COX-2 expression, MKK4 activity, and PI-3 kinase activity induced by UVB was abolished with delphinidin treatment. Collectively, our results demonstrated that delphinidin targets MKK4 and PI-3 kinase directly to suppress COX-2 overexpression, suggesting a potential protective role for delphinin against UVB-mediated skin carcinogenesis.

The cyclin-dependent kinase inhibitor, p21WAF1, promotes angiogenesis by repressing gene transcription of thioredoxin-binding protein 2 in cancer cells

Tue, 22 Sep 2009 07:30:56 PDT

The cyclin-dependent kinase inhibitor, p21^WAF1, induces cell cycle arrest and can act as a tumor suppressor. However, increasing evidence indicates that p21^WAF1 can also increase resistance to some anticancer therapies, and thus promote tumor growth. The mechanisms explaining this paradox have not been explained. We found that conditioned media from MCF-7 breast cancer cells transfected with a p21^WAF1-specific, small interfering RNA (siRNA) significantly reduced endothelial cell migration, invasion and vascular sprouting. Liquid chromatography/mass spectrometry analysis of the conditioned media revealed that p21^WAF1 knockdown significantly reduced secretion of thioredoxin (Trx), a redox protein known to promote tumor angiogenesis. p21^WAF1 knockdown decreased Trx enzymatic activity in cancer cells, by effects on the expression levels of intracellular Trx binding protein-2 (TBP2), known to bind and inactivate Trx. Consistent with these findings, media from cancer cells transfected with TBP2 siRNA promoted endothelial cell invasion, and, blocked the anti-angiogenic effect of p21^WAF1 siRNA. Addition of Trx siRNA blocked the pro-angiogenic effects of TBP2 siRNA. Chromatin immuno-precipitation assays showed p21^WAF1 bound the TBP2 gene promoter. Taken together, our data suggests that p21^WAF1 can induce Trx secretion and angiogenesis in cancer cells, by direct transcriptional repression of the TBP2 promoter.

IL-12-deficiency suppresses 12-O-tetradecanoylphorbol-13-acetate-induced skin tumor development in 7, 12-dimethylbenz(a)anthracene-initiated mouse skin through inhibition of inflammation

Wed, 16 Sep 2009 08:00:05 PDT

Interleukin (IL)-12 deficiency exacerbates tumorigenesis in UV radiation-induced skin. Here, we assessed the effects of IL-12 deficiency on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in 7, 12-dimethylbenz(a)anthracene (DMBA)-initiated mouse skin. Using this two-stage chemical carcinogenesis protocol we found that the development of DMBA/TPA-induced skin tumors was diminished in IL-12p40 knockout (IL-12 KO) mice than in their wild-type counterparts. At the termination of the experiment (at 24 weeks) the skin tumor incidence and tumor multiplicity were significantly lower (p<0.005) in IL-12 KO mice than in their wild-type counterparts, as was the malignant transformation of DMBA/TPA-induced papillomas to carcinomas (p<0.01). Analysis of samples collected at the termination of the experiments for biomarkers of inflammation by immunohistochemical analysis, western blotting, ELISA and real-time PCR, revealed significantly lower levels of COX-2, PGE₂, PCNA, cyclin D1, and the proinflammatory cytokines (TNF-, IL-1β and IL-6) in the DMBA/TPA-treated tumors and tumor-uninvolved skin of IL-12 KO mice than the skin and tumors of DMBA/TPA-treated wild-type mice. Analysis of the skin 6 hours after TPA treatment showed that the TPA-induced promotion of skin edema, inflammatory leukocyte infiltration, COX-2 expression and PGE₂ production was significantly lower in the skin of the IL-12-KO mice than their wild-type counterparts. These results indicate that DMBA/TPA-induced skin tumor development differs from UVB-induced skin tumor development in that endogenous IL-12 acts to inhibit UVB-induced skin tumor development and malignant progression of the skin tumors to carcinoma. In the case of DMBA/TPA-induced skin tumor development, the endogenous IL-12 modulates the tumor promoter stimulation of inflammatory responses.

Selenium modifies the osteoblast inflammatory stress response to bone metastatic breast cancer

Wed, 16 Sep 2009 08:00:04 PDT

Breast cancer frequently metastasizes to the skeleton resulting in bone degradation due to osteoclast activation. Metastases also down regulate differentiation and the bone rebuilding function of osteoblasts. Moreover, cancer cells trigger osteoblast inflammatory stress responses. Pro-inflammatory mediators such as IL-6, MCP-1, COX-2, and iNOS, expressed by osteoblasts (MC3T3-E1) stimulated with human breast cancer cell (MDA-MB-231) conditioned medium, are pivotal to osteoclast activation and metastasis. Given that these genes are regulated by NF-B, a redox- sensitive transcription factor, we hypothesized that selenium (Se) could abrogate the inflammatory response to metastatic breast cancer cells by modulating NF-B. Caffeic acid phenethylether and parthenolide inhibited NF-B activation, as seen by gel-shift assays and immunoblotting for p65 in nuclear fractions, as well as decreased production of IL-6 and MCP-1. Supplementation of MC3T3-E1 with methylseleninic acid (MSA) (500 nM to 4 µM) reduced the activation of NF-B leading to a decrease in IL-6, MCP-1, COX-2 and iNOS in response to MDA-MB-231 conditioned medium. Addition of MSA to osteoblasts for as little as 15 min suppressed activation of NF-B suggesting that short lived active metabolites might be involved. However, brief exposure to MSA also brought about an increase in selenoprotein GPx1. In summary, our data indicate that the osteoblast response to metastatic breast cancer cells is regulated by NF-B activation, which can be effectively suppressed by MSA either through short-lived active metabolites and/or selenoproteins. Thus, Se supplementation may prevent the osteoblast inflammatory response or dampen the viscous cycle established when breast cancer cells, osteoblasts, and osteoclasts interact.

A Polymorphic Variant in Human MDM4 Associates with Accelerated Age of Onset of Estrogen Receptor Negative Breast Cancer

Wed, 16 Sep 2009 23:23:31 PDT

MDM4 (Murine Double Minute 4) shares significant structural homology with MDM2 (Murine Double Minute 2) and interacts and regulates transcriptional activity of the tumor suppressor p53. In tumors with wild-type p53, there is often overexpression of MDM2 or MDM4 leading to functional inactivation of p53. A single nucleotide polymorphism (SNP) in the promoter of human MDM2 (SNP309) was shown to associate with increased MDM2 expression and increased risk of cancer. This study evaluated the association of a SNP in human MDM4 (C>T) with age of onset of breast cancer in two independent cohorts. In cohort one of 675 patients, the average age of diagnosis for women with ER positive and ER negative breast cancers was 53.2 years and 48 years, respectively. In this cohort, homozygous variant (TT) carriers developed ER negative carcinomas at an earlier age than those homozygous wild-type (CC) or heterozygous (TC) such that the age at diagnosis was accelerated by 5.0 years (p = 0.018). This association was validated in a second cohort of breast cancer patients (n = 148), where TT carriers with ER negative cancer developed the disease 3.8 years earlier than CC carriers (p = 0.006). The effect was more pronounced in Caucasians with ER negative ductal carcinomas with TT homozygotes developing disease 7.5 years (p = 0.031) and 6.2 years (p = 7x10^-5) earlier than CC carriers in cohorts 1 and 2, respectively. No association was seen in ER positive ductal cancers suggesting that the SNP in MDM4 only has a functional association in ER negative breast cancer.

Guggulsterone enhances head and neck cancer therapies via inhibition of signal transducer and activator of transcription-3

Wed, 16 Sep 2009 23:23:30 PDT

Treatment of human head and neck squamous cell carcinoma (HNSCC) cell lines with guggulsterone, a widely available, well-tolerated nutraceutical, demonstrated dose-dependent decreases in cell viability with EC₅₀s ranging from 5-8 µM. Guggulsterone induced apoptosis and cell cycle arrest, inhibited invasion and enhanced the efficacy of erlotinib, cetuximab and cisplatin in HNSCC cell lines. Guggulsterone induced decreased expression of both phosphotyrosine and total signal transducer and activator of transcription (STAT)-3, which contributed to guggulsterone's growth inhibitory effect. Hypoxia inducible factor (HIF)-1 was also decreased in response to guggulsterone treatment. In a xenograft model of HNSCC, guggulsterone treatment resulted in increased apoptosis and decreased expression of STAT3. In vivo treatment with a guggulsterone-containing natural product, Guggulipid, resulted in decreased rates of tumor growth and enhancement of cetuximab's activity. Our results suggest that guggulsterone-mediated inhibition of STAT3 and HIF-1 provide a biologic rationale for further clinical investigation of this compound in the treatment of HNSCC.

Plasma membrane calcium ATPase 4 and the remodeling of calcium homeostasis in human colon cancer cells

Tue, 15 Sep 2009 11:25:19 PDT

A remodeling of calcium homeostasis has been identified as a characterizing feature of some cancers. Possible consequences of this include alterations in many pivotal physiological responses including apoptosis, proliferation and gene transcription. An alteration in calcium homeostasis can occur via changes in the expression of proteins that transport calcium and examples of cancers where this is seen includes the prostate and breast. A specific isoform of the calcium efflux pump, plasma membrane Ca²⁺-ATPase 4 (PMCA4), is significantly up-regulated during differentiation of the HT-29 colon cancer cell line suggesting that it may also be altered in colon cancer. We now report that differentiated HT-29 colon cancer cells have pronounced plasma membrane PMCA4 localization, consistent with augmented calcium efflux. Assessment of PMCA4 transcription in human colon cancer samples suggests that PMCA4 is significantly (P < 0.000001) down-regulated early in the progression of some colon cancers as these cells become less differentiated. Inhibition of PMCA4 using siRNA did not induce cell death or augment sensitivity to the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Reversing the colon cancer remodeling of PMCA4 by over-expression reduced cellular proliferation (P < 0.01) and down-regulated transcription of the calcium-sensitive early response gene FOS. Our studies suggest that the remodeling of the calcium signal in colon cancer is associated with compromised calcium efflux at a level that promotes proliferative pathways while avoiding increased sensitivity to apoptotic stimuli.

Activation of the Aryl hydrocarbon receptor pathway enhances cancer cell invasion by up-regulating the MMP expression and is associated with poor prognosis in upper urinary tract urothelial cancer

Tue, 15 Sep 2009 11:25:18 PDT

Aryl hydrocarbon receptor (AhR) and the activation of the AhR pathway are involved in xenobiotic-induced toxicity and carcinogenesis. Although xenobiotics, such as cigarette smoke, contribute to the development of urothelial carcinoma (UC), the relationship between AhR and UC is unclear. In the present study, we investigated AhR expression in 209 patients with upper urinary tract UC. The nuclear expression of AhR was significantly associated with histological grade, pathological T stage, lymphovascular invasion, and lymph node involvement. A multivariate Cox analysis revealed that nuclear AhR expression was a significant and independent predictor for disease-specific survival (hazard ratio = 2.469, P = 0.013). To determine whether the AhR pathway can be activated in the T24 UC cell line, we examined the expression of cytochrome P450 (CYP) 1A1 and CYP1B1, which are target genes of the AhR pathway, following exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand of AhR. TCDD treatment up-regulated the expression levels of AhR, CYP1A1 and CYP1B1. TCDD enhanced T24 cell invasion associated with the up-regulation of matrix metalloproteinase (MMP) -1 and MMP-9. Furthermore, targeting AhR mRNA expression in T24 cells with small interfering RNA (siRNA) down-regulated the mRNA expression of AhR, CYP1A1, CYP1B1, MMP-1, MMP-2 and MMP-9; furthermore, the cells transfected with siRNA for AhR showed decreased invasion activity in comparison to the cells transfected with a non-targeting siRNA. Our results therefore suggest that AhR plays a role in the invasiveness of UC cells and can serve as a marker for the prognosis of upper urinary tract UC.

The Aryl Hydrocarbon Receptor Nuclear Translocator (Arnt) is required for tumor initiation by benzo(a)pyrene

Tue, 15 Sep 2009 11:25:17 PDT

Benzo(a)pyrene (B(a)P) is a ligand for the Aryl Hydrocarbon Receptor (Ahr). After binding ligand, Ahr dimerizes with the Aryl Hydrocarbon Receptor Nuclear Translocator protein (Arnt), and the dimer upregulates the transcription of Cyp1a1, Cyp1b1, and other enzymes involved in the metabolic activation of B(a)P. Arnt null mice die in utero. Mice in which Arnt deletion occurs constitutively in the epidermis die perinatally. In the current study, mice were developed in which the Arnt gene could be deleted specifically in adult skin epidermis. This deletion had no overt pathological effect. Homozygosity for a null NAD(P)H quinone oxidoreductase allele was introduced into the above mouse strain to render it more susceptible to tumor initiation by B(a)P. Deletion of Arnt in the epidermis of this strain completely prevented the induction of skin tumors in a tumor initiation–promotion protocol in which a single topical application of B(a)P acted as the tumor initiating event, and tumor promotion was provided by repeated topical applications of 12-0-tetradecanoyl phorbol-13-acetate, (TPA). In contrast, deletion of Arnt did not prevent the induction of skin tumors in a protocol also using TPA as the promoter, but using as the initiator N-methyl-N’-nitro-N-nitrosoguanidine, whose activity is unlikely to be affected by the activity of Ahr, Arnt or their target genes. These observations demonstrate that Arnt is required for tumor initiation by B(a)P in this system.

Sulindac effects on inflammation and tumorigenesis in the intestine of mice with Apc and Mlh1 mutations

Tue, 15 Sep 2009 11:25:17 PDT

We have previously reported that sulindac, an NSAID, inhibited tumor formation in the small intestine but increased tumors in the colon of Apc^Min/⁺ mice, a model of human familial adenomatous polyposis (FAP). To further explore intestinal regional responses we studied effects of sulindac on additional gene-targeted mouse models of human intestinal tumorigenesis; these were (i) Apc^1638N/+ mouse (chain-termination mutation in exon 15^th of the Apc gene); (ii) Mlh1^+/- mouse (DNA mismatch repair deficiency; a mouse model of human HNPCC; and (iii) double heterozygous Mlh1^+/-Apc^1638N/+ mutant mouse. Mice were fed AIN-76A control diet with or without 0.02% sulindac for 6 months. Intestinal regional tumor incidence, multiplicity, volume and degree of inflammation were used as endpoints. The results showed: (i) sulindac inhibited tumor development in the small intestine of Apc^1638N/+ mice; (ii) in contrast, sulindac increased tumors in the small intestine of Mlh1 mutant mice, a neoplastic effect which persisted in heterozygous compound Mlh1^+/-Apc^1638N/+ mutant mice; (iii) sulindac increased tumors in the cecum of all mice regardless of genetic background; (iv) sulindac decreased inflammation in the small intestine of Apc^1638N/+ mice, but it increased inflammation in the small intestine of Mlh1^+/- mice, and Mlh1^+/-Apc^1638N/+ mice; (v) sulindac enhanced inflammation in the cecum of all mutant mice. Findings indicate that the effects of sulindac in the intestine of these mutant mouse models are likely related to genetic background, and appear to be associated with its inflammatory-inducing response.

Epigenetics in Cancer

Sharma, S., Kelly, T. K., Jones, P. A. — Sun, 13 Sep 2009 21:30:34 PDT

Epigenetic mechanisms are essential for normal development and maintenance of tissue specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Global changes in the epigenetic landscape are a hallmark of cancer. The initiation and progression of cancer, traditionally seen as a genetic disease, is now realized to involve epigenetic abnormalities along with genetic alterations. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer including DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs, specifically microRNA expression. The reversible nature of epigenetic aberrations has led to the emergence of the promising field of epigenetic therapy, which is already making progress with the recent FDA approval of three epigenetic drugs for cancer treatment. In this review, we discuss the current understanding of alterations in the epigenetic landscape that occur in cancer compared to normal cells, the roles of these changes in cancer initiation and progression, including the cancer stem cell model, and the potential use of this knowledge in designing more effective treatment strategies.

Alteration of microRNA expression in vinyl-carbamate-induced mouse lung tumors and modulation by the chemopreventive agent indole-3-carbinol

Melkamu, T., Zhang, X., Tan, J., Zeng, Y., Kassie, F. — Fri, 11 Sep 2009 02:55:52 PDT

MicroRNAs (miRNAs) are small, non-protein-coding RNAs that can function as tumor suppressors or oncogenes. Deregulation of microRNA expression has been reported in lung cancer. However, modulation of miRNA expression by chemopreventive agents remains to be defined. In the present study, we examined if the chemopreventive agent indole-3-carbinol (I3C) reversed vinyl carbamate-induced deregulation of miRNA levels in lung tissues of female A/J mice. Lung tissues were obtained from a previous chemoprevention study, in which mice were treated with VC and given I3C in the diet for 15 weeks. Microarray studies revealed alterations in the expression of a number of miRNAs in lung tumors relative to that of normal lungs. miR-21, mir-31, miR-130a, miR-146b, and miR-377 were consistently up-regulated, whereas miR-1 and miR-143 were down-regulated in lung tumors relative to normal lungs. In mice treated with VC and given I3C in the diet, levels of miR-21, mir-31, miR-130a, miR-146b, and miR-377 were reduced relative to the level in mice treated with the carcinogen only. The results of the microarray study were confirmed by QRT-PCR and gel analysis of PCR products. Further studies with miR21 indicated that PTEN, PDCD4, and RECK are potential targets for the oncogenic effect of miR-21 and the chemopreventive activity of I3C. Taken together, we showed here that miRNAs are deregulated during VC-induced mouse lung tumorigenesis and their levels are modulated by I3C. Therefore, miRNAs and their target genes are promising biomarkers for the diagnosis of lung cancer and efficacy of chemopreventive/chemotherapeutic agents.

Cancer Preventive Activities of Tocopherols and Tocotrienols

Ju, J., Picinich, S. C., Yang, Z., Zhao, Y., Suh, N., Kong, A.-N., Yang, C. S. — Fri, 11 Sep 2009 02:55:52 PDT

The cancer preventive activity of vitamin E has been studied. Whereas some epidemiological studies have suggested a protective effect of vitamin E against cancer formation, many large scale intervention studies with -tocopherol (usually large doses) have not demonstrated a cancer preventive effect. Studies on -tocopherol in animal models also have not demonstrated robust cancer prevention effects. One possible explanation for the lack of demonstrable cancer preventive effects is that high doses of -tocopherol decrease the blood and tissue levels of - and -tocopherols. It has been suggested that -tocopherol, due to its strong anti-inflammatory and other activities, may be the more effective form of vitamin E in cancer prevention. Our recent results have demonstrated that a -tocopherol-rich mixture of tocopherols inhibits colon, prostate, mammary, and lung tumorigenesis in animal models, suggesting that this mixture may have a high potential for applications in the prevention of human cancer. In this review, we discuss biochemical properties of tocopherols, results of possible cancer preventive effects in humans and animal models, and possible mechanisms involved in the inhibition of carcinogenesis. Based on this information, we propose that a -tocopherol-rich mixture of tocopherols is a very promising cancer preventive agent and warrants extensive future research.

Pro-survival of estrogen receptor-negative breast cancer cells is regulated by a BLT2-reactive oxygen species-linked signaling pathway

Choi, J.-A, Lee, J.-W., Kim, H., Kim, E.-Y., Seo, J.-M., Ko, J., Kim, J.-H. — Fri, 11 Sep 2009 02:55:50 PDT

Leukotriene B₄ is an inflammatory mediator with potent biological activities in the pathogenesis of many inflammatory diseases. In the present study, we found that expression of BLT2, a low-affinity leukotriene B₄ receptor, is significantly up-regulated in breast cancer cells. In addition, we observed that inhibition of BLT2 by a specific antagonist, LY255283, or by siBLT2 RNA interference caused dramatic apoptotic cell death in breast cancer cells, especially in the estrogen receptor (ER)-negative MDA-MB-468 and MDA-MB-453 cells, suggesting a role for BLT2 in survival of these breast cancer cells. In an approach to understand the downstream mechanism by which BLT2 mediates the potential pro-survival signaling, we found that the elevated ROS generation is associated with BLT2-mediated survival. Expression of Nox1, a member of the NADPH oxidase family, is also highly up-regulated in a BLT2-dependent manner in these breast cancer cells, suggesting that ‘Nox1-derived ROS’ lie downstream of BLT2. Consistent with the proposed role of ‘Nox1-ROS’ in pro-survival signaling, knock-down of Nox1 with siNox1 or treatment with a ROS scavenging agent caused dramatic apoptotic death in these breast cancer cells. Taken together, our results demonstrate, for the first time, that the ‘BLT2-Nox1-ROS’-linked cascade is involved in the pro-survival signaling, especially in ER-negative breast cancer cells.

Mutational analysis of FANCL, FANCM and the recently identified FANCI suggests that among the 13 known Fanconi Anemia genes, only FANCD1/BRCA2 plays a major role in high-risk breast cancer predisposition

Tue, 08 Sep 2009 06:53:30 PDT

Fanconi Anemia (FA) is a rare recessive syndrome characterized by cellular hypersensitivity to DNA-crosslinking agents. To date 13 FA complementation groups have been described and all 13 genes associated to each of these groups have been currently identified. Three of the known FA genes are also high-risk (FANCD1/BRCA2) or moderate-risk (FANCN/PALB2 and FANCJ/BRIP1) breast cancer susceptibility genes, which makes all members of the FA pathway particularly attractive breast cancer candidate genes. Most FA genes have been screened for mutations in breast cancer families negative for BRCA1/2 mutations but the role of FANCL, FANCM and the recently identified FANCI has not been evaluated to date. This fact and novel data sustaining greater functional relevance of the three genes within the FA pathway prompted us to scrutinize all coding sequences and splicing sites of FANCI, FANCL and FANCM in 95 BRCA1/2-negative index cases from Spanish high-risk breast cancer families. We identified 68 sequence variants of which 24 were coding and 44 non-coding. Six exonic and 26 non-coding variants had not been previously described. None of the coding changes caused clearly pathogenic changes and computational analysis of all non-described intronic variants did not revealed major impact in splicing. With the present study all known FA genes have been evaluated within the context of breast cancer high-risk predisposition. Our results rule out a major role of FANCI, FANCL and FANCM in familial breast cancer susceptibility, suggesting that among the 13 known FA genes, only FANCD1/BRCA2 plays a major role in high-risk breast cancer predisposition.

miR-34a as a prognostic marker of relapse in surgically resected non-small-cell lung cancer

Mon, 07 Sep 2009 07:04:33 PDT

MicroRNAs (miRNAs) have been identified as promising prognostic markers in non-small-cell lung cancer (NSCLC) since they play an important role in oncogenesis. The miR-34 family is composed of three miRNAs (miR-34a, miR-34b, miR-34c) which are part of the p53 network and whose expression is directly induced by p53 in response to DNA damage or oncogenic stress. We have analyzed the impact of miR-34 expression on relapse and overall survival in surgically resected NSCLC patients. For this purpose, we used stem-loop RT-PCR to analyze the expression of the miR-34 family in paired tumor and normal tissue from 70 surgically resected NSCLC patients who received no post-surgical treatment until relapse. In addition, in patients with sufficient tumor tissue, we assessed p53 mutations and the methylation status of the MIRN34A gene promoter region and correlated these findings with miR-34a expression. Molecular findings were correlated with relapse and overall survival. The miR-34 family was downregulated in tumor compared to normal tissue, and low levels of miR-34a expression correlated with a high probability of relapse (p = 0.04). A relation was also found between MIRN34A methylation and miR-34a expression (p = 0.008). Patients with both p53 mutations and low miR-34a levels had the highest probability of relapse (p = 0.001). In the multivariate analysis, miR-34a expression emerged as an independent prognostic marker for relapse. In summary, we have identified miR-34a as a novel prognostic marker in NSCLC patients, providing a potential mechanism for estimating a patient's risk of disease recurrence and a useful tool to help guide treatment decisions.

Endoglin phosphorylation by ALK2 contributes to the regulation of prostate cancer cell migration

Mon, 07 Sep 2009 07:04:32 PDT

Endoglin, a transmembrane glycoprotein that acts as a transforming growth factor beta coreceptor, is down-regulated in PC3-M metastatic prostate cancer cells. When restored, endoglin expression in PC3-M cells inhibits cell migration in vitro, and attenuates the tumorigenicity of PC3-M cells in SCID mice, though the mechanism of endoglin regulation of migration in prostate cancer cells is not known. The current study indicates that endoglin is phosphorylated on cytosolic domain threonine residues by the TGF-β type I receptors ALK2 and ALK5 in prostate cancer cells. Importantly, in the presence of constitutively active ALK2 endoglin did not inhibit cell migration, suggesting that endoglin phosphorylation regulated PC3-M cell migration. Therefore, our results suggest that endoglin phosphorylation is a mechanism with relevant functional consequences in prostate cancer cells. These data demonstrate for the first time that TGF-β receptor-mediated phosphorylation of endoglin is a Smad-independent mechanism involved in the regulation of prostate cancer cell migration.

The intestine-specific transcription factor Cdx2 inhibits {beta}-catenin/TCF transcriptional activity by disrupting the {beta}-catenin/ TCF protein complex

Guo, R.-J., Funakoshi, S., Lee, H. H., Kong, J., Lynch, J. P. — Fri, 04 Sep 2009 05:58:19 PDT

Cdx2 is an intestine-specific transcription factor known to regulate proliferation and differentiation. We previously have reported that Cdx2 limits the proliferation of human colon cancer cells by inhibiting the transcriptional activity of the β-catenin/TCF bipartite complex. Herein we further elucidate this mechanism. Studies with a classic Cdx2 target gene and a canonical Wnt/β-catenin/TCF reporter suggest Cdx2 regulates these promoters by distinctly different processes. Specifically, inhibition of β-catenin/TCF activity by Cdx2 does not require Cdx2 transcriptional activity. Instead, Cdx2 binds β-catenin and disrupts its interaction with the DNA-binding TCF factors, thereby silencing β-catenin/TCF target gene expression. Using Cdx2 mutants, we map the Cdx2 domains required for the inhibition of β-catenin/TCF activity. We identify a subdomain in the N-terminus that is highly conserved and when mutated significantly reduces Cdx2 inhibition of β-catenin/TCF transcriptional activity. Mutation of this subdomain also abrogates Cdx2’s antiproliferative effects in colon cancer cells. In summary, we conclude that Cdx2 binds β-catenin and disrupts the β-catenin/TCF complex. Considering the pivotal role of β-catenin/TCF activity in driving proliferation of normal intestinal epithelial and colon cancer cells, our findings suggest a novel mechanism for Cdx2-mediated regulation of Wnt/β-catenin signaling and cell proliferation.

Resequencing and Copy Number Analysis of the Human Tyrosine Kinase Gene Family in Poorly Differentiated Gastric Cancer

Fri, 04 Sep 2009 05:58:18 PDT

The tyrosine kinase (TK) family is an important regulator of signaling pathways that control a variety of physiological and pathological conditions, and a substantial proportion of TK genes are genetically altered in cancer. To clarify the somatic mutation profile of TK genes and discover potential targets for gastric cancer therapy, we undertook a systematic screening of mutations in the kinase domains of all human TK genes (636 exons of 90 genes) in 17 gastric cancer cell lines and 52 microdissected primary gastric cancers with poorly differentiated histology. We identified 26 non-synonymous alterations (22 genes in total) that included 11 sequence alterations in cell lines and 15 somatic mutations in primary tumors. Recurrent mutations were found in four genes including a known oncogene (NTRK3), the Src kinase family (LTK and CSK) and a potential Wnt signal activator (ROR2). In addition, we analyzed copy number alterations of all the TK gene loci in the same cohort samples by array-based comparative genomic hybridization analysis, and identified 24 high-level amplifications and two homozygous deletions. Both sequence alteration and frequent copy number aberration were detected in two TK genes (HCK and ERBB2), strongly suggesting that they encode potential oncogenes in gastric cancer. Our focused and integrated analyses of systemic resequencing and gene copy number have revealed the novel onco-kinome profile of gastric cancer and pave the way to a comprehensive understanding of the gastric cancer genome.

Plasma protein kinase C (PKC){alpha} as a biomarker for the diagnosis of cancers

Kang, J.-H., Asai, D., Toita, R., Kitazaki, H., Katayama, Y. — Wed, 26 Aug 2009 06:51:16 PDT

Protein kinase C (PKC) plays a key role in the differentiation, proliferation, and apoptosis of cancer cells, and its activity is higher in cancer cells than in normal cells. In the present study, we investigated the existence of activated PKC in plasma and its possibility for cancer diagnosis. Plasma samples were prepared from xenograft mouse models of cancer and from normal mice. Phosphorylation ratios for a PKC-specific peptide substrate (Alphatomega) were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and activated PKC was identified by western blot analysis. Increased levels of activated PKC were found in the plasma of cancer-bearing mice (U87, A549, A431, HuH-7, and B16 melanoma) compared with the levels found in control mice. Phosphorylation ratios for peptide substrate increased with an increase in tumor size. Moreover, the addition of Ro-31-7549, a highly specific inhibitor of PKC, produced a concentration-dependent reduction of phosphorylation ratios, whereas the non-PKC inhibitors, rottlerin and H-89, did not significantly effect phosphorylation ratios. In addition, the level of activated PKC decreased after cancer resection, but increased if the cancer recurred. From these results, we suggest that 1) activated PKC in plasma can be a useful biomarker for the diagnosis of cancers and 2) the level of activated PKC can be monitored to assess the recurrence of cancer after surgical removal. To our knowledge, this is the first report demonstrating the existence of activated PKC in plasma and its possibility for cancer diagnosis.

Pro- and anti-carcinogenic mechanisms of piceatannol are activated dose-dependently in MCF-7 breast cancer cells

Thu, 20 Aug 2009 08:06:10 PDT

Estrogenic pro-carcinogenic effects of piceatannol (PIC) contrast reports about anti-carcinogenic activities of PIC. To explain this contradiction we investigated PIC in estrogen-dependent MCF-7 breast cancer cells and elucidated those cellular mechanisms that correlated with the observed cell effects induced by PIC.

Low PIC concentrations (50 nanomolar) induced c-Myc that depended on progesterone receptor (PR) and estrogen receptor (ER). PR-mediated c-Myc induction by PIC was independent of nuclear PR activity but depended on MAPK signaling and was associated with an acceleration of cancer cell proliferation.

In contrast, 25 µM PIC inhibited dNTP synthesis, activated Chk2 and p38-MAPK and this was accompanied by an attenuation of cancer cell growth. Apoptosis was most likely inhibited due to activation of Akt, however high PIC concentrations (>100 µM) permitted apoptosis-like cell death in consequence to disruption of orchestrated mitotic signaling.

The presented results show for the first time that nanomolar PIC concentrations signal through PR and Erk1/2 and provide a mechanistic explanation why moderate wine consumption – but not other alcoholic beverages– increases the breast cancer risk in women. In contrast, higher PIC concentrations in the micromolar range are considered for adjuvant anticancer therapeutic concepts.

Synbiotic Intervention of Bifidobacterium Lactis and Resistant Starch Protects Against Colorectal Cancer Development in Rats

Le Leu, R. K., Hu, Y., Brown, I. L., Woodman, R. J., Young, G. P. — Thu, 20 Aug 2009 08:06:10 PDT

This study evaluated the effect of a probiotic bacteria ‘Bifidobacterium lactis’, the carbohydrate ‘resistant starch’ (RS) and their combination (synbiotic), on their ability to protect against colorectal cancer. Bifidobacterium lactis has previously been shown to utilise RS as a substrate and up-regulate the acute apoptotic response to a carcinogen in the colon (Le Leu et al. J. Nutr. 135: 996–1001, 2005). Sprague-Dawley rats were divided into 6 equal groups and fed semi-purified diets for 30 weeks. Colonic neoplasms were induced by 2 weekly injections of azoxymethane (15 mg/kg B.W). The experimental groups were as follows: Control - no added dietary fibre or RS; RS in two forms - Hi-maize 958 or Hi-maize 260; Bifidobacterium lactis (lyophilized) - added to control and RS diets (six treatment groups in all). Rats fed RS in combination with Bifidobacterium lactis showed significantly lowered incidence and multiplicity of colonic neoplasms (p<0.01) by over 50% compared to the Control group. There was a trend for protection by RS alone (p=0.07) while no protection against cancer was seen in the group supplemented with only Bifidobacterium lactis. Fermentation events (SCFA, pH) were altered by the inclusion of RS into the diet while the inclusion of Bifidobacterium lactis into the diet had no significant effect on the fermentation parameters. The synbiotic combination of RS and Bifidobacterium lactis significantly protects against the development of colorectal cancer in the rat-azoxymethane model. Synbiotic combination of prebiotic and probiotic seems likely to be a superior preventive strategy to prebiotic alone.

Urinary bladder carcinogenesis induced by chronic exposure to persistent low-dose ionising radiation after Chernobyl accident

Thu, 30 Jul 2009 03:50:42 PDT

Urinary bladder urothelium as well as cells in the microenvironment of lamina propria (endothelial elements, fibroblasts and lymphocytes) demonstrates a number of responses to chronic persistent long-term low-dose ionizing radiation (IR). Thus, oxidative stress occurs, accompanied by up-regulation of at least two signalling pathways (p38 MAPK and NF-kB cascades) and activation of growth factor receptors, in the bladder urothelium of people living in Cesium 137 (¹³⁷Cs) contaminated areas of Ukraine, resulting in chronic inflammation and the development of proliferative atypical cystitis, so-called Chernobyl cystitis, which is considered a possible preneoplastic condition in humans. Furthermore, significant alterations in regulation of cell cycle transitions are associated with increased cell proliferation, along with up-regulated ubiquitination and sumoylation processes as well as inefficient DNA repair (base and nucleotide excision repair pathways) in the affected urothelium. The microenvironmental changes induced by chronic long-term low-dose IR also appear to promote angiogenesis and remodeling of the extracellular matrix that could facilitate invasion as well as progression of pre-existing initiated cells to malignancy. Based on the available findings, new strategies have been developed for predicting and treatment of Chernobyl cystitis - a first step in urinary bladder carcinogenesis in humans.

Beta-carotene affects oxidative stress related DNA damage in lung epithelial cells and in ferret lung.

Tue, 28 Jul 2009 08:04:35 PDT

Beta-carotene (BC) was found to enhance lung cancer risk in smokers. This adverse effect was unexpected because BC was thought to act as an anti-oxidant against cigarette smoke derived radicals. These radicals can directly or indirectly damage DNA, leading to the formation of pro-mutagenic DNA lesions such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-]purin-10(3H)-one deoxyguanosine (M₁dG). Later, it was suggested that high concentrations of BC could also result in pro-oxidant effects. Therefore, we investigated whether high but physiologically feasible concentrations of BC were able to alter i. the formation of radicals in vitro assessed by electron spin resonance spectroscopy (ESR), ii. the levels of 8-oxo-dG and M₁dG in vitro in lung epithelial cells after incubation with H₂O₂ and the smoke derived carcinogen benzo[a]pyrene (B[a]P) and iii. the levels of 8-oxo-dG and M₁dG in vivo in ferrets’ lung after chronic exposure to B[a]P. BC increased in vitro hydroxyl radical formation in the Fenton reaction, but inhibited the formation of carbon centered radicals. Similarly, BC was able to enhance 8-oxo-dG in vitro in lung epithelial cells. On the other hand, BC significantly inhibited M₁dG formation in lung epithelial cells, especially after induction of M₁dG by H₂O₂ or B[a]P. Finally, BC supplementation of ferrets also resulted in a significant decrease in M₁dG, but in contrast to the in vitro experiments, no effect was observed on 8-oxo-dG levels, probably because of increased base excision repair capacities (BER) as assessed by a modified comet assay. These data indicate that the fate of BC being a pro- or anti-oxidant strongly depends on the type of radical involved.

IGFBP7 is a p53 Responsive Gene Specifically Silenced in Colorectal Cancer with CpG Island Methylator Phenotype

Tue, 28 Jul 2009 08:04:36 PDT

A subset of colorectal cancers (CRCs) show simultaneous methylation of multiple genes; these tumors have the CpG island methylator phenotype (CIMP). CRCs with CIMP show a specific pattern of genetic alterations, including a high frequency of BRAF mutations and a low frequency of p53 mutations. We therefore hypothesized that genes inactivated by DNA methylation are involved in the BRAF and p53 signaling pathways. Among those, we examined the epigenetic inactivation of IGFBP7 expression in CRCs. We found that in CRC cell lines, the silencing of IGFBP7 expression was correlated with high levels of DNA methylation and low levels of histone H3K4 methylation. Luciferase and chromatin immunoprecipitation assays in unmethylated cells revealed that p53 induces expression of IGFBP7 upon binding to a p53 response element within intron 1 of the gene. Treating methylated CRC cell lines with 5-aza-2’-deoxycytidine restored p53-induced IGFBP7 expression. Levels of IGFBP7 methylation were also significantly higher in primary CRC specimens than in normal colonic tissue (p<0.001). Methylation of IGFBP7 was correlated with BRAF mutations, an absence of p53 mutations, and the presence of CIMP. Thus epigenetic inactivation of IGFBP7 appears to play a key role in tumorigenesis of CRCs with CIMP by enabling escape from p53-induced senescence.

Interleukin-18 increases metastasis and immune escape of stomach cancer via the down-regulation of CD70 and maintenance of CD44

Tue, 28 Jul 2009 08:04:32 PDT

Cancer cells metastasize to the other site after escaping from the immune system and CD70, CD44 and VEGF play important roles in this process. It is recently reported that interleukin-18 is closely related with the pathogenesis of skin tumor. Therefore, we investigated the role of endogenous IL-18 from stomach cancer on the immune escape mechanism and metastasis via the regulation of CD70, CD44 and VEGF expression. IL-18 and IL-18R expression were not only investigated on tumor tissues (n = 10), and sera (n = 20) from stomach cancer patients, but also on human stomach cancer cell lines. IL-18 and IL-18R expression was found on stomach cancer cell lines and tumor tissues. In addition, IL-18 levels were elevated in sera from cancer patients (P<0.05), compared with sera from normal individuals. Changes in CD70, CD44, and VEGF expression by flow cytometry, immunoblotting and ELISA, and immune susceptibility by ⁵¹Cr-release assay were investigated, after silencing or neutralization of endogenous IL-18. CD70 expression was increased and it increases immune susceptibility of cancer cells. In contrast, CD44 and VEGF expression was decreased and it suppresses neovasculization and the metastasis of stomach cancer. After inoculation of IL-18 siRNA transfected stomach cancer cells into Balb/C (nu/nu) mice, regression of tumor mass was determined by measuring of tumor size. And the number and location of metastatic lesions was investigated by H&E staining. The regression of tumor mass and the suppression of metastasis were observed in the mice, which are injected with IL-18 siRNA transfected cell lines. Our data suggest that endogenous IL-18 might facilitate stomach cancer cell immune escape by suppressing CD70 and increasing metastatic ability by up-regulating CD44 and VEGF.

Inhibition of Vinyl Carbamate-Induced Pulmonary Adenocarcinoma by Indole-3-Carbinol and Myo-inositol in A/J Mice

Wed, 22 Jul 2009 03:46:11 PDT

In previous studies, we reported that indole-3-carbinol (I3C) and myo-inositol (MI) inhibit lung adenoma induced by tobacco smoke carcinogens in A/J mice. In this paper, we extended our work and examined the effects of I3C (70 or 30 µmol/g diet) and MI (56 µmol/g diet) against vinyl carbamate (VC)-induced lung adenocarcinoma by administering the agents from one week after the second of two injections of VC until termination of the study at week 18. The higher dose of I3C decreased multiplicities of tumors on the surface of the lung (26%, P=0.0005), and carcinoma incidence (38%), multiplicity (67%, P < 0.0001), and size (complete abolition of carcinoma with an area of > 1.0 cm²) as well as adenoma with cellular pleomorphism (46%, P < 0.0001). The lower dose of I3C was less effective. MI decreased multiplicities of pulmonary surface tumors (20%, P=0.0005), adenoma with cellular pleomorphism (40%, P < 0.0001), and lung adenoma (52%, P < 0.0001) and the proportion of the biggest carcinoma (carcinoma with an area of > 1.0 cm², P < 0.05). Immunoblot analyses of lung tissues for potential target identification showed that I3C (70 µmol/g diet) inhibits IkappaB degradation, NF-kappaB activation, and expression of Cox-2, phospho-Akt, and fatty acid synthase and activates caspase-3 and PARP cleavage. The effect of MI was limited to inhibition of phospho-Akt and fatty acid synthase expression. Our data show that I3C and MI inhibit lung carcinoma and provide a basis for future evaluation of these compounds in clinical trials as chemopreventive agents for current and former smokers.

Association between global DNA hypomethylation in leukocytes and risk of breast cancer

Tue, 07 Jul 2009 06:32:52 PDT

Background: Global DNA hypomethylation may result in chromosomal instability and oncogene activation, and as a surrogate of systemic methylation activity, may be associated with breast cancer risk.

Methods: Samples and data were obtained from women with incident early-stage breast cancer (I to IIIa) and women who were cancer-free, frequency-matched on age and race. In preliminary analyses, genomic methylation of leukocyte DNA was determined by measuring 5-methyldeoxycytosine (5-mdC), as well as methylation analysis of the LINE-1 repetitive DNA element. Further analyses used only 5-mdC levels. Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (95% CI) for risk of breast cancer in relation to amounts of methylation.

Results: In a subset of samples tested (n = 37), 5-mdC level was not correlated with LINE-1 methylation. 5-mdC level in leukocyte DNA was significantly lower in breast cancer cases than healthy controls (p = 0.001), but no significant case-control differences were observed with LINE-1 methylation (p = 0.176). In the entire data set, we noted significant differences in 5-mdC levels in leukocytes between cases (n = 176) and controls (n = 173); p value<0.001. Compared with women in the highest 5-mdC tertile (T3), women in the second (T2; OR=1.49, 95% CI=0.84-2.65) and lowest tertile (T1; OR=2.86, 95% CI=1.65-4.94) had higher risk of breast cancer (p for trend≤0.001). Among controls only, and cases and controls combined, only alcohol intake was found to be inversely associated with methylation levels.

Conclusion: These findings suggest that leukocyte DNA hypomethylation is independently associated with development of breast cancer.

Leptin Receptor Expression in Middle Eastern Colorectal Cancer and it's Potential Clinical Implication

Thu, 11 Jun 2009 08:55:14 PDT

We investigated the role of leptin receptor(Ob-R) and its relationship with PI3K/AKT activation in colorectal carcinomas(CRC) tissues followed by in vitro studies using a panel of CRC cell lines. Obesity serves an important risk factor of several cancers including CRC which ranks as the second most common cancer in Saudi Arabia. High levels of adipokine leptin and its Ob-R are seen in obesity and also in various carcinomas including CRC. We investigated the proliferative and antiapoptotic effect of leptin on human CRC cell lines Caco-2, HT-29 and SW-840 and the role of PI3K/AKT signaling pathway in mediating these actions. Then the expression of Ob-R and its relationship with clinicopathological features was analyzed in 448 CRC, 229 normal colon mucosa and 24 colorectal adenomas using tissue microarray technology. Treatment with leptin resulted in increased proliferation of CRC cell lines and involved activation of PI3K/AKT signaling pathway. Pretreatment with Ob-R siRNA or PI3K inhibitor inhibited these responses. Ob-R was significantly over expressed in primary CRC relative to adenomas and normal colonic mucosa. In primary CRC, Ob-R significantly correlated with leptin expression, early stage and well differentiated tumors. Intriguingly, patient with Ob-R positive tumors showed significantly better overall survival(p = 0.0098). Leptin plays a critical role in CRC carcinogenesis through PI3K/AKT pathway via Ob-R. Ob-R is a prognostic marker associated with better survival.