Carcinogenesis - Advance Access

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes Inhibit Colon Cancer Cell and Tumor Growth Through Activation of c-Jun N-terminal Kinase

Lei, P., Abdelrahim, M., Cho, S. D., Liu, S., Chintharlapalli, S., Safe, S. — 2008-05-05

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs) activate the orphan receptors peroxisome proliferator-activated receptor (PPAR) and Nur77, and induce receptor-dependent and -independent apoptotic pathways in colon and other cancer cells. Structure-activity studies show that the p-bromo (DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) analogs, which exhibit minimal activation of Nur77 and PPAR, induce expression of CCAAT/enhancer binding protein homologous protein (CHOP/GADD153) in colon cancer cells. Moreover, among a series of bromo and fluoro C-DIM analogs, their induction of CHOP was dependent on the position of the phenyl substituents (para ≥ meta ≥ ortho) and required a free indole group. DIM-C-pPhBr and DIM-C-pPhF not only induced CHOP but also activated death receptor 5 (DR5) (CHOP-dependent), cleavage of caspase 8 and PARP which is consistent with activation of the extrinsic pathway of apoptosis. These responses were associated with activation of c-jun N-terminal kinase (JNK) pathway since inhibition of JNK inhibited induction of the extrinsic apoptotic pathway by these C-DIMs. However, in contrast to classical inducers of endoplasmic reticulum (ER) stress such as tunicamycin and thapsigargin, the C-DIM compounds did not induce glucose-related protein 78 (GRP78) which is a marker of ER stress. Proapoptotic and anticarcinogenic effects were also observed in athymic nude mice bearing RKO cell xenografts and treated with 30 mg/kg/d DIM-C-pPhBr and this was accompanied by increased JNK phosphorylation in the tumors. Thus, the anticarcinogenic activity of DIM-C-pPhBr in colon cancer cells and tumors is related to a novel ER stress-independent activation of JNK.

COX-2/EGFR Expression and Survival among Women with Adenocarcinoma of the Lung

2008-05-02

Previous studies suggest cyclooxygenase-2 (COX-2) expression may predict survival among patients with non-small cell lung cancer (NSCLC). COX-2 may interact with epidermal growth factor receptor (EGFR) suggesting that combined COX-2/EGFR expression may provide predictive value. The extent to which their independent or combined expression is associated with prognosis in women with adenocarcinoma of the lung is unknown. In the present study, we examined relationships between COX-2 expression (n = 238), EGFR expression (n = 158), and dual COX-2/EGFR expression (n = 157) and survival among women with adenocarcinoma of the lung. Overall survival was estimated by constructing Cox proportional hazards models adjusting for other significant variables and stratifying by stage at diagnosis and race. Clinical or demographic parameters were not associated with either COX-2 or EGFR expression. Patients with COX-2 positive tumors tended to have poorer prognosis than did patients with COX-2 negative tumors (HR 1.67; 95% CI 1.01-2.78). African Americans with COX-2 positive tumors had a statistically nonsignificant higher risk of death than African Americans with COX-2 negative tumors (HR 5.58; 95% CI 0.64-48.37). No association between COX-2 expression and survival was observed among Caucasians (HR 1.29; 95% CI 0.72-2.30). EGFR expression was associated with a 44% reduction in the risk of death (HR 0.56; 95% CI 0.32-0.98). COX-2-/EGFR+ tumor expression, but not COX-2+/EGFR+ tumor expression, was associated with survival when compared with other combined expression results. In conclusion, COX-2 and EGFR expression, but not combined COX-2+/EGFR+ expression, independently predict survival of women with adenocarcinoma of the lung.

THE CONTRIBUTION OF ANIMAL-FAT OXIDATION PRODUCTS TO COLON CARCINOGENESIS, THROUGH MODULATION OF TGF-{beta}1 SIGNALING

Biasi, F., Mascia, C., Poli, G. — 2008-05-02

It is now unanimously accepted that neoplastic cells tend to become less susceptible to the growth regulatory effects of TGF-β1, mainly because of reduced expression and/or activity of TGF-β1 specific receptors, as reported for many human cancers including colon cancer. Consequently, a sustained increase of TGF-β1 in the intestinal mucosa, like that caused by inflammatory processes and/or high dietary intake of animal fat, might become crucial for the progression of a neoplastic clone. In fact, this pro-apoptotic and pro-differentiating cytokine could eliminate neoplastic cells still susceptible to TGF-β1’s anti-proliferative action (TGF-β1 receptors positive cells), indirectly favoring the expansion of TGF-β1 resistant ones (TGF-β1 receptors deficient or negative cells). The actual concentration of TGF-β1 in the colonic mucosa undergoing neoplastic transformation is still debated, and the phase of the relevant carcinogenetic process in which a reduced susceptibility to this antiproliferative molecule first occurs has not been precisely established yet. However, no doubt that TGF-β1 level and activity may be up-regulated in cells of the macrophage lineage by animal-fat oxidation products, such as oxysterols and aldehydes, as reviewed here. But, phagocytes as well as fibroblasts, constitutively express TGF-β1, and are accumulating in tumor-associated stroma. Thus, up-regulation of this cytokine system within colonic tumor-associated stroma by excess dietary intake of cholesterol and n-6 polyunsaturated fatty acids appears as a primary mechanism of cancer progression at least in neoplastic lesions of the digestive tract.

TWIST modulates prostate cancer cell-mediated bone cell activity and is up-regulated by osteogenic induction

2008-05-02

TWIST, a helix-loop-helix transcription factor, is highly expressed in many types of human cancer. We have previously found that TWIST confers prostate cancer cells with an enhanced metastatic potential through promoting epithelial-mesenchymal transition and a high TWIST expression in human prostate cancer is associated with an increased metastatic potential. The predilection of prostate cancer cells to metastasize to bone may be due to two interplaying mechanisms, (i) by increasing the rate of bone remodeling, and (ii) by undergoing osteomimicry. We further studied the role of TWIST in promoting prostate cancer to bone metastasis. TWIST expression in PC3, a metastatic prostate cancer cell line, was silenced by siRNA and we found that conditioned medium from PC3 with lower TWIST expression had a lower activity on stimulating osteoclast differentiation and higher activity on stimulating osteoblast mineralization. In addition, we found that these effects were, at least partly, associated with TWIST-induced expression of DKK-1, a factor that promotes osteolytic metastasis. We also examined TWIST and RUNX2 expressions during osteogenic induction of an organ confined prostate cancer cell, 22Rv1. We observed increased TWIST and RUNX2 expressions upon osteogenic induction and down-regulation of TWIST through shRNA reduced the induction level of RUNX2. In summary, our results suggest that, in addition to epithelial-mesenchymal transition, TWIST may also promote prostate cancer to bone metastasis by modulating prostate cancer cell-mediated bone remodeling via regulating the expression of a secretory factor, DKK-1, and enhancing osteomimicry of prostate cancer cells, probably, via RUNX2.

Mechanisms of Malignant Progression

Weinberg, R. A. — 2008-05-02

Abi1 Gene Silencing by Short Hairpin RNA Impairs Bcr-Abl-Induced Cell Adhesion and Migration in vitro and Leukemogenesis in vivo

Yu, W., Sun, X., Clough, N., Cobos, E., Tao, Y., Dai, Z. — 2008-05-02

Abi1 was first identified as the downstream target of Abl tyrosine kinases and was found to be dysregulated in leukemic cells expressing oncogenic Bcr-Abl and v-Abl. Although the accumulating evidence supports a role of Abi1 in actin cytoskeleton remodeling and growth factor/receptor signaling, it is not clear how it contributes to Bcr-Abl-induced leukemogenesis. We show here that Abi1 gene silencing by shRNA attenuated the Bcr-Abl-induced abnormal actin remodeling, MT1-MMP clustering and inhibited cell adhesion and migration on fibronectin-coated surfaces. Although the knockdown of Abi1 expression did not affect growth factor independent growth of Bcr-Abl-transformed Ba/F3 cells in vitro, it impeded competitive expansion of these cells in NOD/SCID mice. Remarkably, the knockdown of Abi1 expression in Bcr-Abl-transformed Ba/F3 cells impaired the leukemogenic potential of these cells in NOD/SCID mice. Abi1 contributes to Bcr-Abl-induced leukemogenesis in part through Src family kinases, as the knockdown of Abi1 expression attenuates Bcr-Abl-stimulated activation of Lyn. Together, these data provide for the first time the direct evidence that supports a critical role of Abi1 pathway in the pathogenesis of Bcr-Abl-induced leukemia

Stage-specific disruption of Stat3 demonstrates a direct requirement during both the initiation and promotion stages of mouse skin tumorigenesis

Kataoka, K., Kim, D. J., Carbajal, S., Clifford, J., DiGiovanni, J. — 2008-05-02

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a variety of human malignancies, and has been suggested to play an important role in carcinogenesis. Recently, our laboratory demonstrated that Stat3 is required for the development of skin tumors via two-stage carcinogenesis using skin-specific loss of function transgenic mice. To investigate further the role of Stat3 in each stage of chemical carcinogenesis in mouse skin, i.e. initiation and promotion stages, we generated inducible Stat3-deficient mice (K5.Cre-ER^T2 x Stat3^fl/fl) that show epidermal specific disruption of Stat3 following topical treatment with 4-hydroxytamoxifen (TM). The epidermis of inducible Stat3-deficient mice treated with TM showed a significant increase in apoptosis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and reduced proliferation following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). In two-stage skin carcinogenesis assays, inducible Stat3-deficient mice treated with TM during the promotion stage showed a significant delay of tumor development and a significantly reduced number of tumors compared with control groups. Inducible Stat3-deficient mice treated with TM before initiation with DMBA also showed a significant delay in tumor development and a significantly reduced number of tumors compared with control groups. Finally, treatment of inducible Stat3-deficient mice that had existing skin tumors generated by the two-stage carcinogenesis protocol with TM (by i.p. injection) led to inhibition of tumor growth compared to tumors formed in control groups. Collectively, these results directly demonstrate that Stat3 is required for skin tumor development during both the initiation and promotion stages of skin carcinogenesis in vivo.

Epigenetic Remodeling During Arsenical-Induced Malignant Transformation

Jensen, T. J., Novak, P., Eblin, K. E., Gandolfi, A. J., Futscher, B. W. — 2008-04-30

Humans are exposed to arsenicals through many routes with the most common being in drinking water. Exposure to arsenic has been associated with an increase in the incidence of cancer of the skin, lung, and bladder. Although the relationship between exposure and carcinogenesis is well documented, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. We evaluated the potential epigenetic component of arsenical action by assessing the histone acetylation state of 13,000 human gene promoters in a cell line model of arsenical-mediated malignant transformation. We show changes in histone H3 acetylation occur during arsenical-induced malignant transformation that are linked to the expression state of the associated gene. DNA hypermethylation was detected in hypoacetylated promoters in the select cases analyzed. These epigenetic changes occurred frequently in the same promoters whether the selection was performed with arsenite [As(III)] or with monomethylarsonous acid [MMA(III)], suggesting that these promoters were targeted in a nonrandom fashion, and likely occur in regions important in arsenical-induced malignant transformation. Taken together, these data suggest that arsenicals may participate in tumorigenesis by altering the epigenetic terrain of select genes.

Interleukin Promoter Polymorphisms and Prognosis in Colorectal Cancer

2008-04-30

There is strong evidence that cancer associated inflammation promotes tumor growth and progression. This is especially true for colorectal cancer (CRC). Interleukins (ILs) are important modulators for inflammation. We examined whether promoter polymorphisms in key IL genes (IL4, IL4R, IL6, IL8, IL10) are associated with the risk or clinical outcome of CRC. Five single nucleotide polymorphisms (SNPs) were analyzed in genomic DNA from a cohort including 308 Swedish incident cases of CRC with data on Dukes’ stage and up to 16 years of follow-up, and 585 healthy controls. The selected SNPs have previously been shown to be functional and/or associated with cancer. None of the analyzed SNPs associated with the risk of CRC. When stratifying by tumor stage, significantly more patients carrying at least one G allele of IL10-1082 had tumors with Dukes’ stages A+B than with stages C+D (p_trend=0.035 for genotype distribution). Analyzing associations with overall survival time, we found the rare T allele of IL4-590 to be related to a longer survival [CT vs. CC Cox proportional hazard ratio (HR) 0.69, 95% confidence intervals (CIs) 0.46-1.03; TT vs. CC 0.32 (0.10-1.03)]. For IL6-174, the CG genotype was associated with a longer survival when compared to the CC genotype [0.64 (0.40-1.01)]. The present study was particularly suitable for survival analysis, because all patients were sampled before the diagnosis of CRC. Our results suggest that the SNPs IL4-590 and IL6-174 may be useful markers for CRC prognosis. The predicted biological effect of these SNPs in relation to promotion of cancer progression is consistent with the observed increased survival time.

Sleep duration, melatonin and breast cancer among Chinese women in Singapore

Wu, A. H., Wang, R., Koh, W.-P., Stanczyk, F. C., Lee, H.-P., Yu, M. C. — 2008-04-30

Background: Sleep duration has been hypothesized to be inversely associated with breast cancer risk, possibly due to greater overall melatonin production in longer sleepers. However, data are inconclusive from the three studies conducted in Western populations on sleep duration and breast cancer risk.

Methods: We investigated the relationship between self-reported usual sleep duration determined at baseline and subsequent risk of breast cancer in the prospective, population-based cohort of the Singapore Chinese Health Study. We excluded from the study women with less than 2 years of follow up due to possible change in sleep pattern among breast cancer cases close to the time of diagnosis. Five hundred and twenty-five incident cases of breast cancer were identified among the remaining 33,528 women after up to 11 years of follow-up.

Results: Among women postmenopausal at baseline, breast cancer risk decreased with increasing sleep duration (P trend = 0.047); those who reported 9+ hours of sleep showed a RR of 0.67 (95% CI = 0.4-1.1) compared to women who reported <=6 hours of sleep. This inverse association was observed primarily in lean women (i.e., body mass index below the median value (23.2 kg/m²)) (P = 0.024). In this study population, irrespective of gender, urinary 6-sulfatoxymelatonin levels increased with increasing self-reported hours of sleep (P trend = 0.035) after adjustment for age and time of day of urine collection. Melatonin levels were 42% higher in those with 9+ vs those with 6 or fewer hours of sleep.

Conclusions: Sleep duration may influence breast cancer risk, possibly via its effect on melatonin levels.

Dietary magnesium and DNA repair capacity as risk factors for lung cancer

2008-04-30

Magnesium (Mg) is required for maintenance of genomic stability; however, data on the relationship between dietary Mg intake and lung cancer are lacking. In an ongoing lung cancer case-control study, we identified 1139 cases and 1210 matched healthy controls with data on both diet and DNA repair capacity (DRC). Dietary intake was assessed using a modified Block-NCI Food Frequency Questionnaire and DRC was measured using the host cell reactivation assay to assess repair in lymphocyte cultures. After adjustment for potential confounding factors including DRC, the odds ratios (OR) and 95% confidence intervals (CI) for lung cancer with increasing quartiles of dietary Mg intake were: 1.0, 0.83 (0.66-1.05), 0.64 (0.50-0.83), 0.47 (0.36-0.61), respectively for all subjects (P-trend<0.0001). Similar results were observed by histology and clinical stage of lung cancer. Low dietary Mg intake was associated with poorer DRC and increased risk of lung cancer. In joint-effects analyses, compared to those with high dietary Mg intake and proficient DRC, the OR (95% CI) for lung cancer in the presence of both low dietary Mg and suboptimal DRC was 2.36 (1.83-3.04). Similar results were observed for men and women. The effects were more pronounced among older subjects (>60 years), current or heavier smokers, drinkers, those with a family history of cancer in first-degree relatives, small cell lung cancer, and late stage disease. These intriguing results need to be confirmed in prospective studies.

A tandem repeat of human telomerase reverse transcriptase (hTERT) and risk of breast cancer development and metastasis in Chinese women

2008-04-15

Telomerase reactivation, which prevents telomere shortening and maintains cell viability, is crucial for the continued growth or progression of cancer cells. A minisatellite tandem repeat, MNS16A, located in the downstream of the human telomerase reverse transcriptase (hTERT) gene was recently identified and reported to have an effect on hTERT expression and telomerase activity. The aim of this study was to test the hypothesis that the MNS16A variant is associated with risk of breast cancer development and metastasis. We genotyped MNS16A variant in hTERT in a case-control study of 1029 histologically confirmed breast cancer patients and 1107 cancer-free controls in Chinese women. The variant genotypes (302/271, 302/243 and 243/243) of MNS16A were associated with a significantly increased risk of breast cancer (adjusted OR = 1.50, 95% CI = 1.15-1.96), compared with the wild-type 302/302 genotype. In stratified analyses, we found that the 302/271 genotype was associated with a significantly increased risk of axillary lymph nodes metastasis (adjusted OR = 2.13, 95% CI = 1.05- 4.33), compared with wild-type 302/302 genotype. These findings indicate that the MNS16A variant in the hTERT gene may contribute to the risk of breast cancer development and metastasis in Chinese women.

Glutathione S-transferase pi mediates proliferation of androgen independent prostate cancer cells.

Hokaiwado, N., Takeshita, F., Naiki-Ito, A., Asamoto, M., Ochiya, T., Shirai, T. — 2008-04-15

Prostate cancers generally acquire an androgen independent growth capacity with progression, resulting in resistance to anti-androgen therapy. Therefore, identification of the genes regulated through this process may be important for understanding of mechanisms of prostate carcinogenesis. We here utilized androgen dependent/independent transplantable tumors, newly established with the "Transgenic Rat Adenocarcinoma in Prostate" (TRAP) model, to analyze their gene expression using microarrays. Among the overexpressed genes in androgen independent prostate cancers compared to the androgen dependent tumors, glutathione S-transferase, pi (GST-pi) was included. In line with this, human prostate cancer cell lines PC3 and DU145 (androgen independent) had higher expression of GST-pi compared to LNCaP (androgen dependent) as determined by semi-quantitative RT-PCR analysis. To investigate roles of GST pi expression in androgen independent human prostate cancers, GST-pi was knocked down by a siRNA, resulting in significant decrease of the proliferation rate in the androgen independent PC3 cell line.

In vivo, administration of GST-pi siRNA/atelocollagen complex decreased GST-pi protein expression, resulting in enhanced numbers of TUNEL positive apoptotic cells. These findings suggest that GST-pi might play important roles in proliferation of androgen independent human prostate cancer cells.

Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2

2008-04-15

Drug resistance during chemotherapy is the major obstacle to the successful treatment of many cancers. Here we report that inhibition of Nrf2 may be a promising strategy to combat chemoresistance. Nrf2 is a critical transcription factor regulating a cellular protective response that defends cells against toxic insults from a broad spectrum of chemicals. Under normal conditions, the low constitutive amount of Nrf2 protein is maintained by the Keap1-mediated ubiquitination and proteasomal degradation system. Upon activation, this Keap1-dependent Nrf2 degradation mechanism is quickly inactivated, resulting in accumulation and activation of the ARE-dependent cytoprotective genes. Since its discovery, Nrf2 has been viewed as a "good" transcription factor that protects us from many diseases. In this study, we demonstrate the dark side of Nrf2: stable overexpression of Nrf2 resulted in enhanced resistance of cancer cells to chemotherapeutic agents including cisplatin, doxorubicin, and etoposide. Inversely, down-regulation of the Nrf2-dependent response by overexpression of Keap1 or transient-transfection of Nrf2-siRNA rendered cancer cells more susceptible to these drugs. Upregulation of Nrf2 by the small chemical tBHQ also enhanced the resistance of cancer cells, indicating the feasibility of using small chemical inhibitors of Nrf2 as adjuvants to chemotherapy to increase the efficacy of chemotherapeutic agents. Furthermore, we provide evidence that the strategy of using Nrf2 inhibitors to increase efficacy of chemotherapeutic agents is not limited to certain cancer types or anti-cancer drugs, thus can be applied during the course of chemotherapy to treat many cancer types.

Lack of DNA mismatch repair protein MSH6 in the rat results in hereditary non-polyposis colorectal cancer-like tumorigenesis

2008-04-15

To understand genetic instability in relation to tumorigenesis experimental animal models have proven very useful. The DNA mismatch repair (MMR) machinery safeguards genomic integrity by repairing mismatches, insertion/deletion loops and responding to genotoxic agents. Here, we describe the functional characterization of a novel rat mutant model in which the mismatch repair gene Msh6 has been genetically inactivated by N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis. This model shows a robust mutator phenotype that is reflected by microsatellite instability and an increased germ line point mutation frequency. Consequently these rats develop a spectrum of tumors with a high similarity to atypical hereditary non-polyposis colorectal cancer in humans. The MSH6 knockout rat complements existing models for studying genetic instable tumorigenesis as it provides experimental opportunities that are not available or suboptimal in current models.

Bioenergetic differences selectively sensitize tumorigenic liver progenitor cells to a new gold(I) compound

2008-04-15

A hallmark of cancer cells is their ability to evade apoptosis and mitochondria play a critical role in this process. Delineating mitochondrial differences between normal and cancer cells has proven challenging due to the lack of matched cell lines. Here we compare two matched liver progenitor cell lines (LPC), one non-tumorigenic (PIL4) the other tumorigenic (PIL2). Analysis of these cell lines and a p53 wild type non-tumorigenic cell line (BMOL) revealed an increase in expression of genes encoding the antiapoptotic proteins cIAP1 and Yap in the PIL2 cells which resulted in an increase in the protein encoded by these genes. PIL2 cells have higher mitochondrial membrane potential (ø_m) compared to PIL4 and BMOL and had greater levels of reactive oxygen species, despite the fact that the mitochondrial antioxidant enzyme, manganese superoxide dismutase, was elevated at transcript and protein levels. Taken together these results may account for the observed resistance of PIL2 cells to apoptotic stimuli compared to PIL4. We tested a new gold compound to show that hyperpolarized ø_m led to its increased accumulation in mitochondria of PIL2 cells. This compound selectively induces apoptosis in PIL2 cells but not in PIL4 nor BMOL. The gold compound depolarized the ø_m, depleted the ATP pool and activated caspase-3 and caspase-9, suggesting apoptosis was mediated via mitochondria. This investigation shows that the non-tumorigenic and tumorigenic LPC are useful models to delineate the role of mitochondrial dysfunction in tumorigenesis and for the future development of mitochondria targeted chemotherapeutics that selectively target tumor cells.

Glypican-3-mediated oncogenesis involves the IGF signaling pathway

2008-04-15

Glypican-3 (GPC3) is the gene responsible for Simpson-Golabi-Behmel overgrowth syndrome. Previously we have shown that GPC3 is overexpressed in hepatocellular carcinoma (HCC). In this study, we further demonstrated the mechanisms for GPC3-mediated oncogenesis. Firstly, GPC3 overexpression in NIH3T3 cells gave to cancer cell phenotypes including growing in serum-free medium and forming colonies in soft agar, or on the other way, GPC3 knockdown in HuH-7 cells decreased oncogenecity. We further demonstrated that GPC3 bound specifically through its N-terminal proline-rich region to both IGF-II and IGF-1R. GPC3 stimulated the phosphorylation of IGF-1R and the downstream signaling molecule ERK in an IGF-II-dependent way. Also, GPC3 knockdown in HCC cells decreased the phosphorylation of both IGF-1R and ERK. Therefore, GPC3 confers oncogenecity through the interaction between IGF-II and its receptor, and the subsequent activation of the IGF signaling pathway. This data is novel to the current understanding of the role of GPC3 in HCC, and will be important in future developments of cancer therapy.

A novel role of thrombospondin-1 in cervical carcinogenesis: Inhibit stroma reaction by inhibiting activated fibroblasts from invading cancer

2008-04-15

Thrombospondin-1 (TSP-1), a potent angiogenesis inhibitor, has been shown to exert different biological functions on various cell types. Here we investigate the role of TSP-1 in tumor stroma reaction, which is mainly characterized by fibroblast activation to create a permissive microenvironment for tumor progression. Immunohistochemistry examinations in the human surgical specimens have shown a downregulation of TSP-1 during the progression of cervical carcinogenesis was accompanied by an emergence in the upregulation of stroma markers, -SMA and desmin. Transfection of SiHa cervical cancer cells with a plasmid expressing the TSP-1 protein exhibited anti-angiogenic activity in vitro, and resulted in reduced tumor growth in SCID mice, which was accompanied by a decrease in tumor vascularization and lower expressions of -SMA and desmin than those in the vector controls. Transfection with TSP-1 and purified TSP-1 added to NIH3T3 cells did not alter the protein levels of -SMA and desmin, but significantly inhibited matrix metalloprotease-2 (MMP-2) activity. Transforming growth factor β (TGF-β), a major factor in the activation of fibroblasts, increased -SMA and desmin expression, and the ability of cell migration and invasion in NIH3T3 cells. The increased migration ability and the invasive ability into tumor cluster of TGF-β-treated-NIH3T3 cells were dose-dependently inhibited by TSP-1. In contrast, ectopic TSP-1 expression in SiHa cells has little effect on the invasive ability of the NIH3T3 cells. Together, our findings demonstrate a novel role of TSP-1 to inhibit tumor stroma reaction that could be attributed to the blockage of activated fibroblasts from invading cancer cells.

Polymorphisms of genes coding for ghrelin and its receptor in relation to anthropometry, circulating levels of IGF-I and IGFBP-3, and breast cancer risk: a case-control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC)

2008-04-11

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, has two major functions: the stimulation of the growth hormone production and the stimulation of food intake. Accumulating evidence also suggests a role of ghrelin in cancer development. We conducted a case-control study on 1359 breast cancer cases and 2389 matched controls, nested within the European Prospective Investigation into Cancer and Nutrition (EPIC), to examine the association of common genetic variants in the genes coding for ghrelin (GHRL) and its receptor (GHSR) with anthropometric measures, circulating insulin growth factor I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3) and breast cancer risk.

Pairwise tagging was used to select the 15 polymorphisms that represent the majority of common genetic variants across the GHRL and GHSR genes.

A significant increase in breast cancer risk was observed in carriers of the GHRL rs171407-G allele (OR: 1.2; 95%CI: 1.0-1.4; P = 0.02). The GHRL SNP rs375577 was associated with a 5% increase in IGF-I levels (P = 0.01). A number of GHRL and GHSR polymorphisms were associated with body-mass-index and height (P between <0.01 and 0.04). The false-positive report probability (FPRP) approach suggests that these results are noteworthy (FPRP<0.20).

The results presented here add to a growing body of evidence that GHRL variations are associated with body-mass-index. Furthermore, we have observed evidence for association of GHRL polymorphisms with circulating IGF-I levels and with breast cancer risk. These associations, however, might also be due to chance findings and further large studies are needed to confirm our results.

Role of FoxO1 activation in MDR1 expression in adriamycin-resistant breast cancer cells

Han, C. Y., Cho, K. B., Choi, H. S., Han, H.-K., Kang, K. W. — 2008-04-04

The development of multidrug resistance can be mediated by a number of different mechanisms but elevated gene expression of MDR1 (P-glycoprotein) has often been a major cause of chemoresistance in many cancer cells. Therefore, the present study aimed to investigate the role of Forkhead box-containing protein, O subfamily (FoxO), transcription factors in regulating the MDR1 gene expression. The proximal promoter region of the human MDR1 contained a putative FoxO binding site, which partially overlapped with the C/EBPβ binding region. Gel shift and immunoblot analysis of subcellular fractions revealed that nuclear levels of FoxO1 and its DNA binding activity were selectively enhanced in MCF-7/ADR cells, which was reversed by a FoxO1 antibody. Reporter gene assays showed that the transcription of MDR1 gene is stimulated by FoxO1 overexpression. Moreover, both MDR1 expression and doxorubicin resistance in MCF-7/ADR cells were reversed by FoxO1 siRNA. The MDR1 expression in MCF-7/ADR cells was also inhibited by insulin, a functional FoxO1 inactivator. In conclusion, FoxO1 is a novel transcriptional activator of MDR1 and is crucial for MDR1 induction in MCF-7/ADR cells.

MDM2 and p53 Polymorphisms are Associated with the Development of Hepatocellular Carcinoma in Patients with Chronic Hepatitis B Virus Infection

2008-04-04

A single nucleotide polymorphism (SNP) in the promoter region of MDM2, SNP 309, is associated with hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus infection. The effect of p53 codon 72 polymorphism Arg72Pro on HCC risk remains inconsistent. This study evaluated the association of MDM2 and p53 polymorphisms with the presence and early onset of HCC in Korean patients with chronic hepatitis B virus (HBV) infection. In total, 583 consecutive patients with chronic HBV infection were classified according to the presence (n = 287) or absence (n = 296) of HCC. The MDM2 SNP 309 and p53 Arg72Pro were genotyped using restriction fragment length polymorphism method. The MDM2 G/G and p53 Pro/Pro genotypes were more frequent in HCC group than in non-HCC group (P < 0.001 and P = 0.004, respectively). Multivariate analysis for the presence of HCC revealed that the odds ratio (OR) for MDM2 G/G over T/T was 4.89 (P < 0.001), and that of p53 Pro/Pro over Arg/Arg was 3.03 (P = 0.006). Combined, MDM2 G/G and p53 Pro/Pro had a synergistic effect on HCC risk, with an OR of 20.78 (P < 0.001). The mean age of tumor onset in patients with MDM2 G/G genotype was 50.9 years, compared to 55.1 with T/T genotype (P = 0.018), and that with p53 Pro/Pro was 49.7 compared to 52.9 with Arg/Arg (P = 0.040). Thus, MDM2 SNP309 and p53 Arg72Pro are associated with the early development of HCC in Korean patients with chronic HBV infection.

Regulation of Hypoxia-inducible Genes by ETS1 Transcription Factor

2008-04-01

HIF-1 regulates the expression of genes that facilitate tumor cell survival by making them more resistant to therapeutic intervention. Recent evidence suggests that the activation of other transcription factors, in cooperation with HIF-1 or acting alone, is involved in the up-regulation of hypoxia-inducible genes. Here we report that high cell density, a condition that might mimic the physiologic situation in growing tumor and most likely representing nutritional starvation, up-regulates hypoxia-inducible genes. This up-regulation can occur in HIF-independent manner since hypoxia-inducible genes CA9, LOXL2, and NDRG1/Cap43 can be up-regulated by increased cell density under both normoxic and hypoxic conditions in both HIF-1-proficient and -deficient mouse fibroblasts. Moreover, cell density up-regulates the same genes in 1HAEo- and A549 human lung epithelial cells. Searching for other transcription factors involved in the regulation of hypoxia-inducible genes by cell density we focused our attention on ETS1. As reported previously, members of ETS family transcription factors participate in the up-regulation of hypoxia-inducible genes. Here we provide evidence that ETS1 protein is up-regulated at high cell density in both human and mouse cells. The involvement of ETS1 in up-regulation of hypoxia-inducible genes was further confirmed in a luciferase reporter assay using co-transfection of ETS1 expression vector with NDRG1/Cap43 promoter construct. The down-regulation of ETS1 expression with siRNA inhibited the up-regulation of CA9 and NDRG1/Cap43 caused by increased cell density. Collectively, our data indicate the involvement of ETS1 along with HIF-1 in regulating hypoxia-inducible genes.

Differentially Expressed Nucleolar TGF-{beta}1 Target (DENTT) exhibits an inhibitory role on tumorigenesis

Kandalaft, L. E., Zudaire, E., Portal-Nunez, S., Cuttitta, F., Jakowlew, S. B. — 2008-04-01

Differentially Expressed Nucleolar TGF-β1 Target (DENTT), also known as Testis-Specific Protein Y-encoded–Like 2 (TSPYL2) and Cell Division Autoantigen-1 (CDA-1), is a member of the TSPY/TSPY-L/SET/NAP-1 protein superfamily. DENTT is expressed in various tissues including normal human lung. Here we investigate the involvement of DENTT in cancer promotion and progression. DENTT mRNA and protein levels were shown to be markedly downregulated in human and mouse primary tumors and in human tumor cell lines. Overexpression of DENTT in human lung (A549-DENTT) and breast (MCF-7-DENTT) cancer cells resulted in diminished growth potential in anchorage-dependent growth assays and reduced capacity to form colonies under anchorage-independent culture conditions. The migratory potential of A549-DENTT and MCF-7-DENTT cells was reduced when compared to empty vector control cells. Treating human lung cell lines with demethylating agents increased DENTT expression significantly. DENTT expression pattern paralleled that of TGF-β1 in normal and malignant tissue and ectopic expression or treatment with TGF-β1 in lung cancer cells was followed by increased DENTT mRNA and protein levels. Collectively, our results suggest a role for DENTT as a suppressor of the tumorigenic phenotype.

Role of ING4 in human melanoma cell migration, invasion, and patient survival

Li, J., Martinka, M., Li, G. — 2008-03-28

ING4 has been reported as a tumor suppressor and shown to diminish colony-forming efficiency, induce p53-dependent apoptosis and arrest cell cycle at G2/M phase. In this study, we investigated the role of ING4 in human melanoma pathogenesis. Using the tissue microarray technology, we found that ING4 expression is significantly decreased in malignant melanoma compared with dysplastic nevi (P < 0.0001, ² test) and reduced ING4 staining is associated with melanoma thickness, ulceration (P = 0.034 and 0.002, respectively, ² test), as well as poor overall and disease-specific 5-year survival in primary melanoma patients (P = 0.0002 and 0.001, respectively, ² test). Cox regression analysis revealed that reduced ING4 staining is an independent factor for the poor prognosis of patients with primary melanomas. Furthermore, we found that overexpression of ING4 suppressed cell migration by 63% and inhibited RhoA activity and Rock-mediated formation of stress fiber in melanoma cells. Moreover, our data showed that overexpression of ING4 inhibited melanoma cell invasion by 43% compared with the control (P = 0.006, t-test) and ING4-overexpressing melanoma cells showed significantly reduced activity of matrix metalloproteinase-2 (MMP-2) and MMP-9. Taken together, this study highlights the importance of ING4 in melanoma pathogenesis and ING4 may serve as a promising prognostic marker and a potential therapeutic target for human melanoma.

Nickel compounds induce phosphorylation of histone H3 at serine 10 by activating JNK-MAPK pathway

Ke, Q., Li, Q., Ellen, T. P., Sun, H., Costa, M. — 2008-03-28

Nickel is a known carcinogen, although the mechanism of its carcinogenicity is not clear. Here, we provide evidence that nickel can induce phosphorylation of histone H3 at its serine 10 residue in a JNK/SAPK-dependent manner. Nickel induces the phosphorylation of c-Jun amino-terminal kinase (JNK), with no effect on the phosphorylation states of the ERK or p38 MAP kinases. An inhibitor of JNK eliminated the nickel-initiated JNK-mediated induction of histone H3 phosphorylation at serine 10, while inhibitors specific for ERK or p38 kinases had no effect on the phosphorylation levels of histone H3 at serine 10 (P-H3S10) in nickel-treated cells. A complete loss of Ni ion induced phosphorylation of H3S10 was observed when JNK was specifically knocked down with RNAi. These results are the first to show the specific JNK-mediated phosphorylation of histone H3 at its serine 10 residue. We show that addition of nickel to an in vitro P-H3S10 dephosphorylation reaction does not change the loss of phosphorylation in the reaction, supporting the notion that nickel causes H3S10 phosphorylation via the JNK/SAPK kinase pathway. It is likely that modification of histone H3 at serine 10 is one of a growing number of epigenetic changes believed to be involved in the carcinogenesis caused by nickel

Modification of the associations between lifestyle, dietary factors and colorectal cancer risk by APC variants

2008-03-28

In a large Scottish case-control study we investigated the effects of APC Asp1822Val (rs459552) and APC Glu1317Gln substitutions on colorectal cancer (CRC) risk and whether these associations were influenced by lifestyle and dietary factors. We did not observe any associations between the variants and CRC risk in the whole population. Post-menopausal women taking hormone replacement therapy (HRT) and participants who consumed a diet low in total fat, saturated fatty acids (SFAs), mono-unsaturated FAs (MUFAs) and trans FAs (tFAs) had a lower risk of CRC (OR (95% CI): 0.53 (0.41, 0.68); 0.84 (0.72, 0.98); 0.72 (0.62, 0.85); 0.85 (0.73, 1.00); 0.78 (0.67, 0.92); respectively). This risk reduction was stronger in those homozygous for the variant APC 1822 allele with significant interaction relationships for HRT, red meat and MUFA intakes (p-for-interaction case-only design: 0.02, 0.002, 0.02; respectively). Low n3 poly-unsaturated FA (PUFA) intake was associated with an increased CRC risk for the wild type and heterozygous APC 1822 individuals but with a decreased CRC risk in those homozygous for the variant allele (p-for-interaction case-only design: 0.09). The interaction relationships with the APC 1317 variant were of the same direction though not significant, possibly due to the low frequency of the variant allele. Our results confirm the findings of three recent case-control studies suggesting a number of possible biological mechanisms. However, further large-scale studies are necessary in order to replicate these findings and confirm the role of these APC gene variants and their interaction with dietary and lifestyle exposures in colorectal carcinogenesis.

Sequence variants of elaC homolog 2 (E. coli) (ELAC2) gene and susceptibility to prostate cancer in the Health Professionals Follow-Up Study

Chen, Y.-C., Giovannucci, E., Kraft, P., Hunter, D. J. — 2008-03-28

Background. Two non-synonymous single nucleotide polymorphisms (SNPs), Ser217Leu and Ala541Thr, in the elaC homolog 2 (E. coli) (ELAC2) gene have been related to prostate cancer risk in previous studies, though with inconsistent results. The association of ELAC2 haplotypes with prostate cancer risk has not yet been explored. We assessed whether sequence variants in ELAC2 were associated with the risk of total or aggressive prostate cancer. Methods. In a nested case-control design within the Health Professionals Follow-Up Study, we identified 659 participants with prostate cancer diagnosed after they provided a blood specimen in 1993 and before January 2000. Controls were 656 age-matched men without prostate cancer who had had a prostate-specific antigen test after providing a blood specimen. We genotyped 8 tagging SNPs in ELAC2 to test for association between sequence variances in ELAC2 and prostate cancer. Results. No individual SNP (including Ser217Leu) was associated with the risk of prostate cancer. Ala541Thr is a rare SNP in this population. One common haplotype (hap4) was statistically significantly associated with an increased risk of prostate cancer [odds ratio (OR) = 1.39, 95% CI = 1.05-1.85)]. Two common promoter SNPs and three common haplotypes were statistically significantly associated with aggressive prostate cancer (carriers vs. non-carriers, snp2: OR = 1.43, snp3: OR = 0.69; hap1: OR = 1.47, hap2: OR = 0.72, hap4: OR = 1.51, global P-value for all common haplotypes = 0.11). Conclusion. Common SNPs and haplotypes of ELAC2 were associated with risk of aggressive prostate cancer

Suppressive effects of nobiletin on hyperleptinemia and colitis-related colon carcinogenesis in male ICR mice

Miyamoto, S., Yasui, Y., Tanaka, T., Ohigashi, H., Murakami, A. — 2008-03-28

Adipocytokines are a group of adipocyte-secreted proteins that have significant effects on the metabolism of lipids and carbohydrates, as well as numerous other processes. A number of recent studies have indicated that some adipocytokines may significantly influence the proliferation of malignant cells in vitro, whereas it remains unclear whether they have similar roles in vivo. In this study, we determined serum levels of adipocytokines in mice with azoxymethane (AOM)- and dextran sulfate sodium (DSS)-induced colon carcinogenesis. Five-week-old ICR mice were given a single intraperitoneal injection of AOM, followed by 1% DSS in drinking water for 7 days. Nobiletin, a citrus flavonoid, was given in the diet (100 ppm) for 17 weeks. Thereafter, the incidence and number of colon tumors, and serum concentration of adipocytokines were determined at the end of week 20. The serum leptin level in AOM/DSS-treated mice was 6 times higher than that in untreated mice, whereas there were no significant differences in the levels of triglycerides, adiponectin, and IL-6. Feeding with nobiletin abolished colonic malignancy and notably decreased the serum leptin level by 75%. Further, nobiletin suppressed the leptin-dependent, but not -independent, proliferation of HT-29 colon cancer cells and decreased leptin secretion through inactivation of mitogen-activate protein kinase/ extracellular signaling-regulated kinase (MEK), but not that of adiponectin in differentiated 3T3-L1 mouse adipocytes in a dose-dependent manner. Taken together, our results suggest that higher levels of leptin in serum promote colon carcinogenesis in mice, while nobiletin has chemopreventive effects against colon carcinogenesis, partly through regulation of leptin levels

Polymorphism of genes related to insulin sensitivity and the risk of biliary tract cancer and biliary stone: a population-based case-control study in Shanghai, China

2008-03-28

Biliary tract cancer, encompassing tumors of the gallbladder, extrahepatic bile ducts, and ampulla of Vater, is a rare but highly fatal malignancy. Obesity and gallstones, both related to insulin resistance, are linked to an elevated risk of biliary cancer. The peroxisome proliferator-activated receptors (PPARs) and the retinoid X receptors (RXRs), expressed in adipose tissue, play a key role in the regulation of obesity-related insulin sensitivity, thus genetic variants of these two receptor genes may be related to biliary cancer and stones. We examined the associations of seven single nucleotide polymorphisms (SNPs) in the PPAR-, PPAR-, RXR-, RXR-β and INS genes with biliary cancer and stones in a population-based case-control study in Shanghai, China. We included 237 gallbladder, 127 extrahepatic bile duct, and 47 ampulla of Vater cancer cases, 895 stone cases, and 786 population controls. Relative to individuals with the RXR-β C51T (rs2076310) CC genotype, those having the TT genotype had a 1.6-fold risk for bile duct cancer (OR = 1.67; 95% CI = 0.99-2.84), with a more pronounced association among men (OR = 2.30; 95% CI = 1.14-4.65; P interaction = 0.07). This marker was also associated with a higher risk of gallstones among subjects with a higher body mass index (BMI) (≥23kg/m²) (OR = 1.80; 95% CI = 1.09-2.94), although the interaction with BMI was not statistically significant (P interaction = 0.28). No association was found between other variants and biliary cancers and stones. Results from this population-based study suggest that certain genetic variants involved in the regulation of obesity-related insulin sensitivity may increase susceptibility to bile duct cancer and gallstones

Notch-1 Regulates Transcription of the Epidermal Growth Factor Receptor Through p53

2008-03-20

The Notch pathway plays key roles in development and is increasingly recognized for its importance in cancer. We previously demonstrated over-expression of Notch-1 and its ligands in gliomas and showed their knockdown inhibits glioma cell proliferation and survival. To elucidate mechanisms downstream of Notch-1 in glioma cells, we performed microarray profiling of glioma cells transfected with Notch-1 siRNA. Notable among down-regulated transcripts was the epidermal growth factor receptor (EGFR), known to be over-expressed or amplified in gliomas and prominent in other cancers as well. Further studies confirmed that Notch-1 inhibition decreased EGFR mRNA and EGFR protein in glioma and other cell lines. Transfection with Notch-1 increased EGFR expression. Additionally, we found a significant correlation in levels of EGFR and Notch-1 mRNA in primary high-grade human gliomas. Subsequent experiments showed that p53, an activator of the EGFR promoter, is regulated by Notch-1. Experiments with p53-positive and -null cell lines confirmed that p53 partially mediates the effects of Notch-1 on EGFR expression. These results show for the first time that Notch-1 up-regulates EGFR expression and also demonstrate Notch-1 regulation of p53 in gliomas. These observations have significant implications for understanding the mechanisms of Notch in cancer and development.

Fisetin, a novel dietary flavonoid causes apoptosis and cell-cycle arrest in human prostate cancer LNCaP cells

Khan, N., Afaq, F., Syed, D. N., Mukhtar, H. — 2008-03-20

Novel dietary agents for prevention and therapy of prostate cancer are desired. The aim of this study was to determine the effect of fisetin, a tetrahydroxyflavone, on inhibition of cell-growth and induction of apoptosis in human prostate cancer cells. Treatment of fisetin (10-60 µM; 48 h) was found to result in a decrease in the viability of LNCaP, CWR22R1 and PC-3 cells but had only minimal effects on normal prostate epithelial PrEC cells as assessed by MTT assay. Treatment of LNCaP cells with fisetin also resulted in G1-phase arrest which was associated with a marked decrease in the protein expression of cyclin D1, D2 and E and their activating partner cdk2, 4 and 6 with concomitant induction of WAF1/p21 and KIP1/p27. Fisetin treatment also resulted in induction of apoptosis, PARP cleavage, modulation in the expressions of Bcl2-family proteins, inhibition of PI3K and phosphorylation of Akt at Ser⁴⁷³ and Thr³⁰⁸. There was also induction of mitochondrial release of cytochrome c into cytosol, downregulation of XIAP and upregulation of Smac/DIABLO on treatment of cells with fisetin. Treatment of cells with fisetin also resulted in significant activation of caspase-3, -8 and -9. Pretreatment of cells with caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced activation of caspases. These data provides the first evidence that fisetin could be developed as an agent against prostate cancer.

The Aryl Hydrocarbon Receptor (AhR) Inhibits Vanadate-Induced Vascular Endothelial Growth Factor (VEGF) Production in TRAMP Prostates

Fritz, W. A., Lin, T.-M., Peterson, R. E. — 2008-03-20

Hypoxia inducible factor-1 (HIF-1) and aryl hydrocarbon receptor nuclear translocator (ARNT) are PAS family transcription factors. During angiogenesis and tumor growth, HIF-1 dimerizes with ARNT, inducing expression of many genes, including vascular endothelial growth factor (VEGF). ARNT also dimerizes with the aryl hydrocarbon receptor (AhR). AhR null (Ahr^-/-) transgenic adenocarcinoma of the mouse prostate (TRAMP) mice develop prostate tumors with greater frequency than AhR wild-type (Ahr^+/+) TRAMP mice, even though prevalence of prostate epithelial hyperplasia is not inhibited. This suggests that Ahr inhibits prostate carcinogenesis. In TRAMP mice, prostatic epithelial hyperplasia results in stabilized HIF-1, inducing expression of VEGF, a prerequisite for tumor growth and angiogenesis. Since ARNT is a common dimerization partner of AhR and HIF-1, we hypothesized that the AhR inhibits prostate tumor formation by competing with HIF-1 for ARNT, thereby limiting VEGF production. Prostates from Ahr^+/+, Ahr^+/- and Ahr^-/- C57BL/6J TRAMP mice were cultured in the presence of graded concentrations of vanadate, an inducer of VEGF through the HIF-1/ARNT pathway. Vanadate induced VEGF protein in a dose-dependent fashion in Ahr^+/- and Ahr^-/- TRAMP prostate cultures, but not in Ahr^+/+ cultures. However, vanadate induced upstream proteins in the phosphatidylinositol 3-kinase signaling cascade to a similar extent in TRAMP prostates of each Ahr genotype, evidenced by Akt phosphorylation. These findings suggest that AhR sequesters ARNT, decreasing interaction with HIF-1 reducing VEGF production. Since VEGF is required for tumor vascularization and growth, these studies further suggest that reduction in VEGF correlates with inhibited prostate carcinogenesis in Ahr^+/+ TRAMP mice.

Epigenetic inactivation of the Secreted frizzled-related protein-5 (SFRP5) gene in human breast cancer is associated with unfavorable prognosis

2008-03-19

Disruption of the Wnt pathway is thought to be crucial in the development of human cancer. Pathway inhibitory members of the secreted frizzled-related protein (SFRP)-family were found to be downregulated due to epigenetic inactivation in various malignancies. To date, only SFRP1 has been studied in human breast cancer and we questioned whether other SFRP genes may be implicated in the pathogenesis of this disease as well. An initial realtime PCR analysis of SFRP5 expression in normal human tissues (n=9) revealed weak expression in most tissues, including breast. Malignant mammary cell lines showed further SFRP5 expression loss in five of six cases. Consistently, in matched pairs of primary breast tumor/normal breast tissue this downregulation (>5-fold) could be confirmed (n=8/13; 62%). We identified promoter methylation as the predominant mechanism of SFRP5 gene silencing, since SFRP5 promoter methylation correlated significantly with loss of SFRP5 expression in cell lines (P=0.040) and primary tumors (P=0.003). Moreover, cancerous cell lines re-expressed SFRP5 mRNA following treatment with DNA-demethylating drugs. Of 168 primary breast carcinomas, 73% harbored a methylated SFRP5 promoter while 27% were unaffected by epigenetic alteration. Most interestingly, SFRP5 methylation was associated with reduced overall survival (OS) (P=0.045) and was an independent risk factor affecting OS in a multivariate Cox proportional hazard model (HR: 4.55; 95%CI: 1.01-20.56; P=0.049). In conclusion, SFRP5 is a target of epigenetic inactivation in human breast cancer, supporting the hypothesis of its role as tumor suppressor gene. SFRP5 methylation may be a novel DNA-based biomarker potentially useful in clinical breast cancer management.

Chromosomal Aberration Frequency in Lymphocytes Predicts the Risk of Cancer: Results from a Pooled Cohort Study of 22,358 Subjects in 11 Countries

2008-03-19

Mechanistic evidence linking chromosomal aberration (CA) to early stages of cancer has been recently supported by the results of epidemiological studies which associated CA frequency in peripheral lymphocytes of healthy individuals to future cancer incidence. To overcome the limitations of single studies and to evaluate the strength of this association a pooled analysis was carried out. The pooled database included 11 national cohorts and a total of 22,358 cancer free individuals who underwent genetic screening with CA for biomonitoring purposes during 1965–2002 and were followed up for cancer incidence and/or mortality for an average of 10.1 years; 368 cancer deaths and 675 incident cancer cases were observed. Subjects were classified within each laboratory according to tertiles of CA frequency. The relative risk (RR) of cancer was increased for subjects in the medium (RR = 1.31; 95% confidence interval (CI) 1.07–1.60) and in the high (RR = 1.41; 95% CI 1.16–1.72) tertiles, when compared with the low tertile. This increase was mostly driven by chromosome-type aberrations. The presence of ring chromosomes increased the RR to 2.22 (95% CI= 1.34-3.68). The strongest association was found for stomach cancer (RR_medium=1.17 (95% CI= 0.37-3.70); RR_high=3.13 (95% CI= 1.17-8.39)). Exposure to carcinogens did not modify the effect of CA levels on overall cancer risk. These results reinforce the evidence of a link between CA frequency and cancer risk and provide novel information on the role of aberration subclass and cancer type.

Genetic variations of microRNAs in human cancer and their effects on the expression of miRNAs

2008-03-19

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level to lead to mRNA degradation or repressed protein production. The expression of miRNA is deregulated in many types of cancers. To determine whether genetic alterations in miRNA genes are associated with cancers, we have systematically screened sequence variations in several hundred human miRNAs from over 100 human tumor tissues and 20 cancer cell lines. We identified 8 new single nucleotide polymorphisms (SNPs) and 14 novel mutations (or very rare SNPs) that specifically present in human cancers. These mutations/SNPs are distributed in the regions of pri-, pre- and even mature miRNAs, respectively. Importantly, while most of the mutations did not exert detectable effects on miRNA function, a G->A mutation at 19 nt downstream of miRNA let-7e led to a significant reduction of its expression in vivo, indicating that miRNA mutation could contribute to tumorigenesis. These data suggest that further screening for genetic variations in miRNA genes from a wide variety of human cancers should increase the discovery and identification of molecular diagnostic and therapeutic targets and complement the mutation analysis of consensus coding sequences in human cancers.

DNA adducts and cancer risk in prospective studies: a pooled analysis and a meta-analysis

2008-03-14

Background - Bulky DNA adducts are biomarkers of exposure to aromatic compounds, and of the ability of the individual to metabolically activate carcinogens and to repair DNA damage. Their ability to predict cancer onset is uncertain.

Methods - We have performed a pooled analysis of three prospective studies on cancer risk in which bulky DNA adducts have been measured in blood samples collected from healthy subjects (N = 1947; average follow-up 51 to 137 months). In addition, we have performed a meta-analysis by identifying all articles on the same subject published up to the end of 2006, including case-control studies.

Results - In the pooled analysis a weakly statistically significant increase in the risk of lung cancer was apparent (14% per unit standard deviation change in adduct levels, 95% Confidence Interval, 1 to 28%; using the WMD (weighted mean difference) method, 0.15 standard deviation units higher adducts in cases then in controls). The association was evident only in current smokers, and was absent in former smokers. Also the meta-analysis, which included both lung and bladder cancers, showed a statistically significant association in current smokers, while the results in never smokers were equivocal; in former smokers no association was detected.

Conclusion - The results of our pooled and meta-analyses suggest that bulky DNA adducts are associated with lung cancer arising in current smokers after a follow-up of several years.

Associations of dietary methyl donor intake with MLH1 promoter hypermethylation and related molecular phenotypes in sporadic colorectal cancer

2008-03-13

Intake of dietary factors that serve as methyl group donors may influence promoter hypermethylation in colorectal carcinogenesis. We investigated whether dietary folate, vitamins B2 and B6, methionine and alcohol were associated with MLH1 hypermethylation, and the related molecular phenotypes of MLH1 protein expression, microsatellite instability (MSI) and BRAF mutations in patients with colorectal carcinomas.

Within the Netherlands Cohort Study on diet and cancer (n=120,852), 648 cases (367 men and 281 women) and 4,059 subcohort members were available for data analyses from a follow-up period between 2.3 and 7.3 years after baseline. Gender-specific adjusted incidence rate ratios (RR) were calculated over categories of dietary intake in case-cohort analyses.

The intakes of folate, vitamin B2, methionine and alcohol were not associated with risk of tumors showing MLH1 hypermethylation, those lacking MLH1 protein expression, or with MSI. Among men, we observed strong positive associations between folate and BRAF-mutated tumors (RR=3.04, for the highest versus lowest tertile of intake, P_trend=0.03), and between vitamin B6 and tumors showing MLH1 hypermethylation (highest vs. lowest tertile: RR=3.23, P_trend=0.03). Among women, the relative risks of tumors with BRAF mutations or MLH1 hypermethylation were also increased in the highest tertiles of folate and vitamin B6 intake respectively, but these did not reach statistical significance.

The positive associations between folate intake and tumors harboring BRAF mutations and between vitamin B6 intake and those showing MLH1 hypermethylation were most pronounced among men, and may suggest that these vitamins enhance CRC risk through genetic as well as epigenetic aberrations.

Plasma levels of carotenoids, retinol, and tocopherol and the risk of gastric cancer in Japan: A nested case-control study

2008-03-13

Fruits and vegetables have been suggested to confer protection against diseases such as cancer through the effects of antioxidants, often represented by carotenoids. We investigated the impact of carotenoids, retinol and tocopherol on gastric cancer development in a large nested case-control study among Japanese with known Helicobacter pylori infection status. A total of 36 745 subjects aged 40 to 69 in the Japan Public Health Center-based Prospective Study who responded to the baseline questionnaire and provided blood samples in 1990-1995 were followed until 2004. Plasma levels of carotenoids in 511 gastric cancer cases and 511 matched controls were measured by high performance liquid chromatography. Odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were estimated using conditional logistic regression models. Plasma level of β-carotene was inversely associated with the risk of gastric cancer (compared with the lowest quartile: OR=0.63, 95% CI, 0.31 to 0.75; OR=0.48, 95% CI, 0.31 to 0.75 and OR=0.46, 95% CI, 0.28 to 0.75, for quartile 2, 3 and 4 respectively, P_trend < .01). Inverse associations were evident in men for -carotene (P_trend = .04) and β-carotene (P_trend < .01), but not in women, who had relatively higher plasma levels compared to men. We found no statistically significant association between plasma levels of lutein/zeaxanthin, lycopene, retinol, - or -tocopherol and gastric cancer risk. Our findings suggest that those who have very low plasma levels of -carotene and β-carotene are at a higher risk of gastric cancer.

Maternal and offspring genetic variants of AKR1C3 and the risk of childhood leukemia

2008-03-13

The Aldo-keto reductase 1C3 (AKR1C3) gene located on chromosome 10p15-p14, a regulator of myeloid cell proliferation and differentiation, represents an important candidate gene for studying human carcinogenesis. In a prospectively enrolled population-based case-control study of Han Chinese conducted in Kaohsiung in southern Taiwan, a total of 114 leukemia cases and 221 controls less than 20 years old were recruited between November 1997 and December 2005. The present study set out to evaluate the association between childhood leukemia and both maternal and offspring's genotypes. To do so, we conducted a systematic assessment of common SNPs at the 5' flanking 10k bp to 3'UTR of AKR1C3 gene. Gln5His and three tagSNPs (rs2245191, rs10508293, and rs3209896) and 1 multi-marker (rs2245191, rs10508293, and rs3209896) were selected with average 90% coverage of un-tagged SNPs by using the HapMap II dataset. Odds ratios (OR) and 95% confidence intervals (CI) were adjusted for age and gender. After correcting for multiple comparisons, we observed that risk of developing childhood leukemia is significantly associated with rs10508293 polymorphism on intron 4 of the AKR1C3 gene in both offspring alone and in the combined maternal and offspring genotypes (nominal p<0.0001, permutation p<0.005). The maternal methylenetetrahydrofolate reductase (MTHFR) A1298C polymorphism was found to be an effect modifier of the maternal intron 4 polymorphism of the AKR1C3 gene (rs10508293) and the childhood leukemia risk. In conclusion, this study suggests that AKR1C3 polymorphisms may be important predictive markers for childhood leukemia susceptibility.

Delphinidin, a dietary anthocyanidin, inhibits platelet derived growth factor ligand/receptor (PDGF/PDGFR) signaling

2008-03-13

Most cancers are dependent on the growth of tumor blood vessels and inhibition of tumor angiogenesis may thus provide an efficient strategy to retard or block tumor growth. Recently, tumor vascular targeting has expanded to include not only endothelial cells (EC), but also the smooth muscle cells (SMC) which contribute to a mature and functional vasculature. We have previously reported that delphinidin, a major biologically active constituent of berries, inhibits the vascular endothelial growth factor (VEGF)-induced phosphorylation of VEGF receptor-2 (VEGFR-2) and blocks angiogenesis in vitro and in vivo. In the present study, we show that delphinidin also inhibits activation of the platelet derived growth factor-BB (PDGF-BB) receptor-β (PDGFR-β) in SMC and that this inhibition may contribute to its antitumor effect. The inhibitory effect of delphinidin on PDGFR-ß was very rapid and led to the inhibition of PDGF-BB-induced activation of ERK-1/2 signaling and of the chemotactic motility of SMC, as well as the differentiation and stabilization of EC and SMC into capillary-like tubular structures in a 3D co-culture system. Using an anthocyan-rich extract of berries, we show that berry extracts were able to suppress the synergistic induction of vessel formation by basic fibroblast growth factor-2 and PDGF-BB in the mouse Matrigel plug assay. Oral administration of the berry extract also significantly retarded tumor growth in a lung carcinoma xenograft model. Taken together, these results provide new insight into the molecular mechanisms underlying the antiangiogenic activity of delphinidin that will be helpful for the development of dietary-based chemopreventive strategies.

Aberrations of chromosome 19 in asbestos-associated lung cancer and in asbestos-induced micronuclei of bronchial epithelial cells in vitro

2008-03-13

Exposure to asbestos is known to induce lung cancer, and our previous studies have suggested that specific chromosomal regions, such as 19p13, are preferentially aberrant in lung tumours of asbestos-exposed patients. Here, we further examined the association between the 19p region and exposure to asbestos, using array comparative genomic hybridization and fluorescence in situ hybridization (FISH) in lung tumours and FISH characterization of asbestos-induced micronuclei in human bronchial epithelial BEAS 2B cells in vitro. We detected an increased number of 19p losses in the tumours of asbestos-exposed patients in comparison with tumours from non-exposed subjects with similar distribution of tumour histology in both groups (13/33; 39% vs. 3/25; 12%, P=0.04). In BEAS 2B cells, a 48-h exposure to crocidolite asbestos (2.0 µg/cm²) was found to induce centromere-negative micronuclei harbouring chromosomal fragments. Furthermore, an increased frequency of rare micronuclei containing a 19p fragment was observed after the crocidolite treatment in comparison with untreated controls (6/6000 vs. 1/10000, P=0.01). The results suggest that 19p has significance in asbestos-associated carcinogenesis and that asbestos may be capable of inducing specific chromosome aberrations.

Hypoxia-inducible factor (HIF)-1{alpha} directly enhances the transcriptional activity of stem cell factor (SCF) in response to hypoxia and epidermal growth factor (EGF)

2008-03-13

Stem cell factor (SCF) plays important roles in tumor growth and angiogenesis. However, its regulatory mechanism remains largely undefined. Here we report that hypoxia up-regulated the expression of SCF in MCF-7 breast cancer cells in both mRNA and protein levels. When HIF-1 expression was knocked down by RNA interference, the SCF cells expression of SCF was decreased significantly. Furthermore, the SCF receptor, c-kit phosphorylation was significantly strengthened by the condition culture media (CCM) from hypoxic MCF-7 and MCF-7-c cells. The survival of A549 cells was more dependent on SCF under hypoxia. Analysis of SCF promoter 5’-flanking region revealed a potential hypoxia-response element (HRE; 5’-GCGTG-3’) located at -68 to -64 relative to the transcriptional start site. Chromatin immunoprecipitation (ChIP) assay demonstrated that HIF-1 directly bound to this region under normoxia, and this binding activity was significantly enhanced under hypoxia. Overexpression of HIF-1 significantly up-regulated the expression of luciferase reporter gene under control of the SCF promoters in both MCF-7 cells and HEK-293 cells, but mutation of the HRE site completely blocked this effect. EGF was also able to enhance the SCF expression under normoxia in MCF-7 cells, which was dependent on HIF-1. Taken together, our data demonstrated that HIF-1 was a key regulator of SCF expression in breast cancer cells. Hypoxia and EGFR signal co-existed in the tumor microenvironment and might promote angiogenesis through HIF-1-mediated up-regulation of SCF and other angiogenic factors.

t10,c12-conjugated linoleic acid stimulates mammary tumor progression in her2/erbB2 mice through activation of both proliferative and survival pathways

Meng, X., Shoemaker, S., McGee, S. O., Ip, M. M. — 2008-03-13

The t10,c12 isomer of conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis, metastasis from a transplantable mouse mammary tumor, and angiogenesis; however it stimulates mammary tumorigenesis in transgenic mice overexpressing ErbB2 in the mammary epithelium (ErbB2 transgenic mice). In the current study, we report that a 4 week supplementation of the diet with 0.5% t10,c12-CLA stimulated the growth of established ErbB2-overexpressing mammary tumors by 30%, and increased the number of new tumors from 11 to 82%. Additionally, when t10,c12-CLA supplementation of ErbB2 transgenic mice was initiated at 21 weeks of age, a time just prior to tumor appearance, overall survival was decreased from 46.4 weeks in the control to 39.0 weeks in the CLA group, and survival after detection of a palpable tumor from 7.5 weeks to 4.6 weeks. Short-term supplementation from 10-14 or 21-25 weeks of age temporarily accelerated tumor development, but over the long-term, there was no significant effect on mammary tumorigenesis. Long-term as well as a short 4 week supplementation increased mammary epithelial hyperplasia and lobular development, and altered the mammary stroma; this was reversible in mice returned to the control diet. t10,c12-CLA altered proliferation and apoptosis of the mammary epithelium, although this differed depending on the length of administration and/or the age of the mice. The increased tumor development with t10,c12-CLA was associated with increased phosphorylation of the IGF-I/insulin receptor, as well as increased signaling through the MEK/ERK and PI3K/Akt pathways; however, neither phospho-ErbB2, nor ErbB2, were altered.

A distinct ERCC1 haplotype is associated with mRNA expression levels in prostate cancer patients

2008-03-10

Both genetic variants and mRNA expression of DNA repair and tumor suppressor genes have been investigated as molecular markers for therapy outcome. However, the phenotypic impact of genetic variants often remained unclear, thus the rationale of their use in risk prediction may be limited. We therefore analyzed genetic variants together with anthropometric and lifestyle factors to see how these affect mRNA levels of ERCC1, MDM2 and TP53 in primary blood lymphocytes. mRNA expression was measured in 376 prostate cancer patients by quantitative real-time PCR after reverse transcription, and ERCC1 rs11615 T>C, ERCC1 rs3212986 C>A, MDM2 rs2279744 T>G and TP53 rs17878362 (p53PIN3) polymorphisms were determined. Considerable inter-individual differences in mRNA expression were found (coefficients of variation: ERCC1, 45%; MDM2, 43%; TP53, 35%). ERCC1 expression was positively correlated with plasma levels of β-carotene (p = 0.03), and negatively correlated with canthaxanthin (p = 0.02), and lutein (p = 0.02). Overall, the polymorphisms affected mRNA expression only weakly. Carriers of a distinct ERCC1 haplotype (CC) showed, however, significantly lower expression values than non-carriers (p = 0.001). Applying logistic regression, we found that CC haplotype carriers had a 1.69-fold increased odds ratio (95% confidence interval 1.06-2.71) for reduced ERCC1 mRNA levels. This low ERCC1 expression might be associated with reduced DNA repair and better therapy response. In summary, the association we have found between ERCC1 genotype and mRNA expression supports recent clinical observations that genetic variation in ERCC1 can affect treatment outcome and prognosis. Our study further revealed a modulating effect by nutritional factors.

Genomic Analysis Suggests Higher Susceptibility Of Children To Air Pollution

2008-03-10

Differences in biological responses to exposure to hazardous airborne substances between children and adults have been reported, suggesting children to be more susceptible. Aim of this study was to improve our understanding of differences in susceptibility in cancer risk associated with air pollution by comparing genome-wide gene expression profiles in peripheral blood of children and their parents. Gene expression analysis was performed in blood from children and parents living in two different regions in the Czech Republic, with different levels of air pollution. Data were analyzed by two different approaches; one method first selected significantly differentially expressed genes and analyzed these gene lists for overrepresented biological processes, while the other applied the T-profiler tool to directly perform pathway analyses on the total gene set without pre-selection of significantly modulated gene expressions. In addition, gene expressions in both children and adults were investigated for associations with micronuclei frequencies. Both analysis approaches returned considerably more genes or gene groups and pathways that significantly differed between children from both regions than between parents. Very little overlap was observed between children and adults. The two most important biological processes or molecular functions significantly modulated in children, but not in adults, are nucleosome and immune response related. Our study suggests differences between children and adults in relation to air pollution exposure at the transcriptome level. The findings underline the necessity of implementing environmental health policy measures specifically for protecting children's health.

Excision Repair is Required for Genotoxin-Induced Mutagenesis in Mammalian Cells

2008-03-10

Certain hexavalent chromium [Cr(VI)] compounds are human lung carcinogens. Although much is known about Cr-induced DNA damage, very little is known about mechanisms of Cr(VI) mutagenesis and the role that DNA repair plays in this process. Our goal was to investigate the role of excision repair pathways in Cr(VI)-mediated mutagenesis in mammalian cells. Repair-proficient Chinese hamster ovary (CHO) cells (AA8), nucleotide excision repair (NER)-deficient (UV-5) and base excision repair (BER)-inhibited cells were treated with Cr(VI) and monitored for forward mutation frequency at the HPRT locus. BER was inhibited using methoxyamine hydrochloride (Mx) which binds to AP sites generated during BER. Notably, we found that both NER-deficient (UV-5, UV-41) and BER-inhibited (AA8 + Mx) cells displayed attenuated Cr(VI) mutagenesis. To determine whether this was unique to Cr(VI), we included the alkylating agent, methylmethane sulfonate (MMS) and ultraviolet radiation (260 nm) in our studies. Similar to Cr(VI), UV-5 cells exhibited a marked attenuation of MMS mutagenesis, but were hypermutagenic following UV exposure. Moreover, UV-5 cells expressing human XPD displayed similar sensitivity to Cr(VI) and MMS induced mutagenesis as AA8 controls indicating that the genetic loss of NER was responsible for attenuated mutagenesis. Interestingly, Cr(VI)-induced clastogenesis was also attenuated in NER-deficient and BER-inhibited cells. Taken together, our results suggest that NER and BER are required for Cr(VI) and MMS-induced genomic instability. We postulate that, in the absence of excision repair, DNA damage is channeled into an error-free system of DNA repair or damage tolerance.

Dietary curcumin modulates transcriptional regulator(s) of phase I and phase II enzymes in benzo(a)pyrene-treated mice: mechanism of its anti-initiating action

Garg, R., Gupta, S., Maru, G. — 2008-03-04

Curcumin has been shown to possess anti-initiating and anti-promoting activity in experimental systems. However, the mechanism(s) of its action(s) are not fully elucidated in vivo. In the present study mechanism(s) of curcumin-mediated anti-initiation were investigated in mice employing benzo(a)pyrene (B[a])P) as a model carcinogen. Dietary pre-treatment of mice with chemopreventive doses of curcumin showed significant inhibition of B(a)P-induced enzyme activity, protein and mRNA levels of cytochrome P450 1A1/1A2 in liver and lungs. Although curcumin alone did not alter the basal levels of aryl hydrocarbon receptor (AhR), it significantly decreased the B(a)P-induced AhR protein levels, its phosphorylation, nuclear translocation and subsequent binding to DNA thereby decreasing the transactivation of CYP1A. Dietary curcumin led to increase in NF-E2-related factor-2 (Nrf2) protein levels and enhanced its nuclear translocation in liver and lungs of mice as compared to controls. Additionally, increased binding of Nrf2 to antioxidant response element (ARE) occurred in nuclear extracts from liver and lungs of mice pre-treated with dietary curcumin. Induction of activity, protein and mRNA levels of glutathione-S-transferase (GST), its isoforms and NAD(P)H:quinone oxidoreductase-1 (NQO1) by dietary curcumin in mice paralleled the curcumin-mediated activation of Nrf2, leading to increased detoxification of B(a)P. In agreement with the observed curcumin-mediated decrease in B(a)P-induced phase I enzyme and concomitant induction of phase II enzymes, pre-treatment with dietary curcumin resulted in significant reduction of B(a)P-induced DNA adduct, oxidative damage and inflammation. To conclude, curcumin exhibits anti-initiating effects via modulating the transcriptional regulator(s) of phase I and phase II enzymes in mice.

Arsenite alters global histone H3 methylation

Zhou, X., Sun, H., Ellen, T. P., Chen, H., Costa, M. — 2008-03-04

Arsenic (As) is a well-characterized human carcinogen but is generally not mutagenic. The evidence that arsenic induces both loss of global DNA methylation and gene promoter DNA hypermethylation have suggested that epigenetic mechanisms may play an important role in arsenic induced carcinogenesis. In the present study, we examined the change in histone methylation by arsenic exposure. In human lung carcinoma A549 cells, exposure to inorganic trivalent arsenic (arsenite) increased H3K9 di-methylation (H3K9me2) and decreased H3K27 tri-methylation (H3K27me3), both of which represent gene silencing marks, while increasing the global levels of the H3K4 tri-methylation (H3K4me3), a gene activating mark. The increase in H3K9me2 was mediated by an increase in the histone methyltransferase G9a protein and mRNA levels. We also observed strikingly significant altered histone modifications induced by very low dose (0.1 µM) arsenite. Taken together, these results suggest a potential mechanism by which arsenic induces carcinogenesis through the alteration of specific histone methylations that represent both gene silencing and activating marks. Furthermore, these marks are known to affect DNA methylation, and it is likely that arsenic's effect is not limited to histone modifications alone, but extends, perhaps by them, to DNA methylations as well. Future studies in our laboratory will address the genomic location of these silencing and activating marks using ChIP-on-chip technology.

Cytoplasmic RASSF2A is a Pro-apoptotic Mediator Whose Expression is Epigenetically Silenced in Gastric Cancer

2008-02-29

Gastric cancer cells often show altered Ras signaling, though the underlying molecular mechanism is not fully understood. We examined the expression profile of eight RASSF genes plus MST1/2 and found that RASSF2A is the most frequently downregulated in gastric cancer. RASSF2A was completely silenced in six of ten gastric cancer cell lines as a result of promoter methylation, and expression was restored by treating the cells with 5-aza-2’-deoxycytidine. Introduction of RASSF2A into non-expressing cell lines suppressed colony formation and induced apoptosis. These effects were associated with the cytoplasmic localization of RASSF2A and morphological changes to the cells. cDNA microarray analysis revealed that RASSF2A suppresses expression of inflammatory cytokines, which may in turn suppress angiogenesis and invasion. In primary gastric cancers, aberrant methylation of RASSF2A was detected in 23 of 78 (29.5%) cases, and methylation correlated significantly with an absence of the lymphatic invasion, absence of venous invasion, absence of lymph node metastasis, less advanced stages, Epstein-Barr virus, absence of p53 mutations and the presence of the CpG island methylator phenotype-high. These results suggest that epigenetic inactivation of RASSF2A is required for tumorigenesis in a subset of gastric cancers.