Thymosin beta-4(Tβ4), actin-sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase(GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK-3 activity is regulated by Tβ4 and it is associated with Tβ4-mediated migration in gastric cancer cells. The level of Tβ4 expression was various in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU 668, was dependent on a relative expression level of Tβ4. Cell migration was higher in SNU668 with a higher expression level of Tβ4 than that in SNU638 with a lower Tβ4. While the level of phospho-GSK-3α(inactive), β-catenin, E-cadherin, and E-cadherin:β-catenin complex was relatively higher, phospho-GSK-3β(inactive) was lower in SNU638 as compared to those in SNU668 cells. LiCl, GSK-3α/β inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference(si)-RNA of GSK-3α increased SNU638 cell migration in accordance with the reduction of E-cadherin:β-catenin complex formation through a decrease in β-catenin and E-cadherin. Expression level of GSK-3α/β, β-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tβ4-siRNA and by the treatment with acetylated(Ac)-SDKP, tetrapeptide of Tβ4, respectively. E-cadherin expression in SNU638 cells was decreased by β-catenin-siRNA. PD98059, MEK inhibitor, or U0126, Erk inhibitor, reduced SNU668 cell migration accompanying an increase in phospho-GSK-3α, β-catenin and E-cadherin. Taken together, data indicated that the expression of GSK-3α, β-catenin and E-cadherin could be negatively regulated by Tβ4-induced phospho-Erk. It suggests that Tβ4 could be a novel regulator to control Wnt signaling pathways. © 2012 Wiley-Liss, Inc.

Previously we have identified a panel of breast cancer anti-estrogen resistance (BCAR) genes. Several of these genes have clinical relevance because mRNA or protein levels associate with tamoxifen resistance or tumor aggressiveness. We postulated that changes in activation status of protein signaling networks induced by BCAR genes may provide better insight into the mechanisms underlying anti-estrogen resistance. Key signal transduction pathways were analyzed for changes in activation or expression using reverse-phase protein microarrays probed with 78 antibodies against signaling proteins with known roles in tumorigenesis. We used ZR-75-1-derived cell lines transduced with AKT1, AKT2, BCAR1, BCAR3, BCAR4, EGFR, GRB7, HRAS, HRAS^v12, or HEF1 and MCF7-derived cell lines transduced with BCAR3, BCAR4, or EGFR. In the anti-estrogen-resistant cell lines we observed increased phosphorylation of several pathways involved in cell proliferation and survival. All tamoxifen-resistant cell lines contained high levels of phosphorylated AKT and its biochemically linked substrates FOXO1/3. The activation of ERBB2, ERBB3 and the downstream modulators FAK and SHC were activated in cells with overexpression of BCAR4. Remarkable differences were observed for the levels of activated AMPK alpha1, cyclins, STAT5, STAT6, ERK1/2 and BCL2. The comparison of the cell signaling networks in estrogen-dependent and -independent cell lines revealed biochemically linked kinase-substrate markers that comprised systemically activated signaling pathways involved in tamoxifen resistance. Our results show that this model provides insights into the molecular and cellular mechanisms of breast cancer progression and anti-estrogen resistance. This knowledge may help the development of novel targeted treatments. © 2012 Wiley-Liss, Inc.

Cigarette smoke (CS) and dietary factors play a major role in cancer epidemiology. At the same time, however, the diet is the richest source of anticancer agents. Berries possess a broad array of health protective properties and were found to attenuate the yield of tumors induced by individual carcinogens in the rodent digestive tract and mammary gland but failed to prevent lung tumors induced by typical CS components in mice. We exposed whole-body Swiss ICR mice to mainstream CS, starting at birth and continuing daily for 4 months. Aqueous extracts of black chokeberry and strawberry were given as the only source of drinking water, starting after weaning and continuing for 7 months, thus mimicking an intervention in current smokers. In the absence of berries, CS caused a loss of body weight, induced early cytogenetical damage in circulating erythrocytes and histopathological alterations in lung (emphysema, blood vessel proliferation, alveolar epithelial hyperplasia, and adenomas), liver (parenchymal degeneration), and urinary bladder (epithelial hyperplasia). Both berry extracts inhibited the CS-related body weight loss, cytogenetical damage, liver degeneration, pulmonary emphysema, and lung adenomas. Protective effects were more pronounced in female mice, which may be ascribed to modulation by berry components of the metabolism of estrogens implicated in lung carcinogenesis. Interestingly, both the carcinogen and the chemopreventive agents tested are complex mixtures that contain a multitude of components working through composite mechanisms.arette smoke (CS) and dietary factors play a major role in cancer epidemiology. At the same time, however, the diet is the richest source of anticancer agents. Berries possess a broad array of health protective properties and were found to attenuate the yield of tumors induced by individual carcinogens in the rodent digestive tract and mammary gland but failed to prevent lung tumors induced by typical CS components in mice. We exposed whole-body Swiss ICR mice to mainstream CS, starting at birth and continuing daily for 4 months. Aqueous extracts of black chokeberry and strawberry were given as the only source of drinking water, starting after weaning and continuing for 7 months, thus mimicking an intervention in current smokers. In the absence of berries, CS caused a loss of body weight, induced early cytogenetical damage in circulating erythrocytes and histopathological alterations in lung (emphysema, blood vessel proliferation, alveolar epithelial hyperplasia, and adenomas), liver (parenchymal degeneration), and urinary bladder (epithelial hyperplasia). Both berry extracts inhibited the CS-related body weight loss, cytogenetical damage, liver degeneration, pulmonary emphysema, and lung adenomas. Protective effects were more pronounced in female mice, which may be ascribed to modulation by berry components of the metabolism of estrogens implicated in lung carcinogenesis. Interestingly, both the carcinogen and the chemopreventive agents tested are complex mixtures that contain a multitude of components working through composite mechanisms. © 2012 Wiley-Liss, Inc.

Treatment of advanced stage cervical cancers with (chemo)radiation causes cytotoxicity through induction of high levels of DNA damage. Tumour cells respond to DNA damage by activation of the ‘DNA damage response’ (DDR), which induces DNA repair and may counteract chemoradiation efficacy. Here, we investigated DDR components as potential therapeutic targets and verified the predictive and prognostic value of DDR activation in cervical cancer patients treated with (chemo)radiation.

In a panel of cervical cancer cell lines, inactivation of ATM or its substrate 53BP1 clearly gave rise to cell cycle defects in response to irradiation. Concordantly, clonogenic survival analysis revealed that ATM inhibition, but not 53BP1 depletion, strongly radio-sensitised cervical cancer cells. In contrast, ATM inhibition did not radiosensitise non-transformed epithelial cells or non-transformed BJ fibroblasts. Interestingly, high levels of active ATM prior to irradiation were related with increased radioresistance.

To test whether active ATM in tumours prior to treatment also resulted in resistance to therapy, immunohistochemistry was performed on tumour material of advanced stage cervical cancer patients (n=375), treated with (chemo)radiation. High levels of phosphorylated (p-)ATM (P=0.006, HR=1.817) were related to poor loco-regional disease-free survival. Furthermore, high p-ATM levels predicted shorter disease-specific survival (P=0.038, HR=1.418). Presence of p-53BP1 was associated with p-ATM (P=0.001, OR=2.206), but was not related to any clinicopathological features or survival.

In conclusion, both our in vitro and patient-related findings indicate a protective role for ATM in response to (chemo)radiation in cervical cancer and point at ATM inhibition as a possible means to improve efficacy of (chemo)radiation. © 2012 Wiley-Liss, Inc.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive form of cancer with a tendency to invade surrounding healthy tissues, leading to a largely incurable disease. Despite many advances in modern medicine, there is still a lack of early biomarkers as well as efficient therapeutical strategies. The melastatin-related transient receptor potential 7 channel (TRPM7) is a non-selective cation channel which is involved in maintaining Ca²⁺ and Mg²⁺ homeostasis. It has been recently reported to regulate cell differentiation, proliferation and migration. However, the role of TRPM7 in PDAC progression is far to be understood. In the present study, we show that TRPM7 is 13-fold overexpressed in cancer tissues compared to the healthy ones. Furthermore, TRPM7 staining is stronger in tumors with high grade, suggesting a correlation between TRPM7 expression and PDAC progression. Importantly, TRPM7 expression is inversely related to patient survival. In BxPC-3 cell line, dialyzing the cytoplasm during the patch-clamp whole-cell recording with a 0-Mg²⁺ solution activated a non-selective current with a strong outward rectification. This cation current is inhibited by intracellular Mg²⁺ and by TRPM7 silencing. The downregulation of TRPM7 by siRNA dramatically inhibited intracellular Mg²⁺ fluorescence and cell migration without affecting cell proliferation suggesting that TRPM7 contributes to Mg²⁺ entry and cell migration. Moreover, external Mg²⁺ following TRPM7 silencing fully restored the cell migration. In summary, our results indicate that TRPM7 is involved in the BxPC-3 cell migration via a Mg²⁺-dependent mechanism and may be a potential biomarker of poor prognosis of PDAC. © 2012 Wiley-Liss, Inc.

Insulin growth factor-I receptor (IGF-IR) is expressed in mesothelioma and therefore an attractive target for therapy. The anti-tumor activity of cixutumumab, a humanized monoclonal antibody to IGF-IR, in mesothelioma and relationship to IGF-IR expression was investigated using eight early passage tumor cells obtained from patients, nine established cell lines and an in vivo human mesothelioma tumor xenograft model. Although IGF-IR expression at the mRNA and protein level was present in all mesothelioma cells, using a quantitative ELISA immunoassay there was considerable variability of IGF-IR expression ranging from 1 to 14 ng/mg of lysate. Using flow cytometry the number of IGF-IR surface receptors varied from >2,000 to 50,000 sites/cell. Cells expressing >10,000 sites/cell had greater than 10% growth inhibition when treated with cixutumumab (100 μg/mL). Cixutumumab also induced antibody-dependent cell-mediated toxicity (ADCC) (>10% specific lysis) in cell lines, which had >20,000 IGF-IR sites/cell. Treatment with cixutumumab decreased phosphorylation of IGF-IR, Akt and Erk in cell lines, H226 and H28 having 24,000 and 51,000 IGF-IR sites/cell respectively but not in the cell line H2052 with 3,000 IGF-IR sites/cell. In vivo, cixutumumab treatment delayed growth of H226 mesothelioma tumor xenografts in mice and improved the overall survival of these mice compared to mice treated with saline (p< 0.004). Our results demonstrate that the anti-tumor efficacy of cixutumumab including inhibition of IGF-IR downstream signaling is highly correlated with IGF-IR sites/cell. A phase II clinical trial of cixutumumab is currently ongoing for treatment of patients with mesothelioma. © 2012 Wiley-Liss, Inc.

Invasiveness is one of the key features of aggressive prostate cancer, however our understanding of the precise mechanisms effecting invasion remains limited. The ceramide hydrolyzing enzyme acid ceramidase (AC), overexpressed in most prostate tumors, causes an aggressive and invasive phenotype through downstream effectors that have not yet been well characterized. Here we demonstrate that AC, through generation of sphingosine-1-phosphate (S1P), promotes Ets1 nuclear expression and binding to the promoter region of matrix-degrading protease cathepsin B. Through confocal microscopy and flow cytometry, we found that AC overexpression promotes pericellular localization of cathepsin B and its translocation to the outer leaflet of the cell membrane. AC overexpressing cells have an increased abundance of cathepsin B-enriched invasive structures and enhanced ability to invade through a collagen matrix, but not in the presence of an inhibitor of cathepsin B. In human prostate tissues, AC and cathepsin B overexpression were strongly associated and may relate to poor cancer outcome. These results demonstrate a novel pathway by which AC, through S1P, promotes an invasive phenotype in prostate cancer by causing overexpression and secretion of cathepsin B through activation and nuclear expression of Ets1. As prostate cancer prognosis is dramatically worse when invasion has occurred, this study provides critical insight into the progression towards lethal prostate cancer.

A diet high in linoleic acid (an ω-6 PUFA) increased the formation of miscoding etheno (ε) - DNA adducts in WBC-DNA of women, but not in men (Nair et al, 1997). This gender specificity could result from an interaction between ω-6 PUFA intake and estrogen catabolism, via redox-cycling of 4-hydroxyestradiol (4-OH-E₂) and subsequent lipid peroxidation (LPO).

In this study, we investigated whether in premenopausal women LPO-derived adducts in WBC-DNA are affected by serum concentration of 2- and 4-hydroxyestradiol metabolites and by fatty acid intake. DNA extracted from buffy coat and plasma samples, both blindly coded from healthy women (N = 124, median age 40 yrs) participating in the EPICHeidelberg cohort study were analyzed. Three LPO-derived exocyclic DNA adducts, εdA, εdC and M₁dG were quantified by immuno-enriched ³²P-postlabelling and estradiol metabolites by ultra-sensitive GC-mass spectrometry.

Mean M₁dG levels in WBC-DNA were distinctly higher than those of εdA and εdC, and all were positively and significantly interrelated. Serum levels of 4-OH-E₂, but not of 2-OHE₂, metabolites were positively related to etheno DNA adduct formation. Positive correlations existed between M₁dG levels and linoleic acid intake or the ratios of dietary linoleic acid/oleic acid and PUFA/MUFA. Aerobic incubation of 4-OH-E₂, arachidonic acid and calf thymus DNA yielded 2 - 3 fold higher etheno DNA adduct levels when compared to assays containing 2-OH-E₂ instead.

In conclusion, this study is the first to compare M₁dG and etheno-DNA adducts and serum estradiol metabolites in human samples in the same subjects. Our results support a novel mechanistic link between estradiol catabolism, dietary ω-6 fatty acid intake, and LPOinduced DNA damage supported by an in vitro model. Similar studies in human breast epithelial tissue and on amplification of DNA-damage in breast cancer patients are in progress. © 2012 Wiley-Liss, Inc.

Pancreatic cancer (PC) is the most aggressive malignant disease, ranks as the fourth most leading cause of cancer related death among men and women in the United States. We present here that plumbagin (PL), a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L, inhibits the growth of PC cells both in vitro and in vivo model systems. PL treatment induces apoptosis and inhibits cell viability of PC cells (PANC1, BxPC3, and ASPC1). In addition, i.p administration of PL (2 mg/kg body weight, 5 days a week) in SCID mice beginning 3 days after ectopic implantation of PANC1 cells resulted in a significant (P<0.01) inhibition of both tumor weight and volume. PL treatment inhibited 1) constitutive expression of EGFR, pStat3Tyr705, pStat3Ser727, 2) DNA binding of Stat3, and 3) physical interaction of EGFR with Stat3, in both cultured PANC1 cells and their xenograft tumors. PL treatment also inhibited phosphorylation and DNA-binding activity of NF-κB in both cultured PC cells (PANC1, ASPC1) and in PANC1 cells xenograft tumors. Downstream target genes (cyclin D1, MMP9, and Survivin) of Stat3 and NF-κB were similarly inhibited. These results suggest that PL may be used as a novel therapeutic agent against human PC. © 2012 Wiley-Liss, Inc.

Though tobacco smoking is the primary risk factor for lung cancer, a significant fraction of lung cancer deaths occur in lifetime non-smokers. In this paper, we calculate the burden of lung cancer in never-smokers attributable to previously identified risk factors in North America, Europe, and China, using population-based estimates of exposure prevalence and estimates of relative risk derived from recently published meta-analyses. Population attributable fractions (PAFs) for individual risk factors ranged from 0.40% to 19.93%. Due to differences in the prevalence of exposures, the PAFs associated with several of the risk factors varied greatly by geographical region. Exposure to the selected risk factors appeared to explain a much larger proportion of lung cancer cases in never-smokers in China than in Europe and North America. Our results demonstrate the geographic variability of the epidemiology of lung cancer in never-smokers, and highlight the need for further research in this area, particularly in Europe and North America. © 2012 Wiley-Liss, Inc.

Melanoma is an increasingly common and potentially fatal malignancy of the skin and some mucous membranes. Since no cure exists for metastatic disease, there is an urgent need for novel drugs. 2′-Deoxy-5-fluorouridylyl-(3′-5′)-3′-C-ethynylcytidine [5-FdU(3′-5′)ECyd] and 3′-C-ethynylcytidinylyl-(5′-->1-O)-2-O-octadecyl-sn-glycerylyl-(3-O-->5′)-2′-deoxy-5-fluorouridine [ECyd-lipid-5-FdU] represent cytostatic active duplex drugs, that can be metabolized into various active antimetabolites. We evaluated the cytotoxicity of these heterodinucleoside phosphate analogues, their corresponding monomers ECyd and 5-FdU and combinations thereof on six metastatic melanoma cell lines and six ex-vivo patient-derived melanoma cells in comparison to current standard cytostatic agents and the BRAF V600E inhibitor Vemurafenib. In vitro (real-time)-proliferation assays demonstrated that 5-FdU(3′-5′)ECyd and ECyd-lipid-5-FdU had a high cytotoxic efficacy causing 75% melanoma cell death at concentrations in the nano- and micromolar range. Cytotoxicity was conducted by induction of DNA cleavage indicating apoptotic cells. Chicken embryotoxicity demonstrated that the duplex drugs were less toxic than 5-FdU at 0.01 µM. In vivo the duplex drug 5-FdU(3′-5′)ECyd was efficacious in the murine LOX IMVI melanoma xenograph model upon administration of 11.2 mg/kg/injection every fourth day. Both duplex drugs are promising novel cytostatic agents for the treatment of malignant melanoma meriting clinical evaluation. © 2012 Wiley-Liss, Inc.

Integrins play a role in tumor growth and metastasis. However, the effect of integrin inhibition has not been visualized on single cancer cells in vivo. In this study, we used a powerful subcellular in vivo imaging model to demonstrate how an anti-integrin antibody affects seeding and growth of osteosarcoma cells on the lung. The 143B human osteosarcoma cell line expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus was established. Such double-labeled cells enable imaging of apoptosis and mitosis and other nuclear-cytoplasmic dynamics. Using the double-labeled osteosarcoma cells, single cancer-cell seeding in the lung after i.v. injection of osteosarcoma cells was imaged. The anti-β1 integrin monoclonal antibody, AIIB2, greatly inhibited the seeding of cancer cells on the lung while a control antibody had no effect. To image the efficacy of the anti-integrin antibody on spontaneous metastasis, mice with orthotopically-growing 143B-RFP cells in the tibia were also treated with AIIB2 or control anti-rat IgG1 antibody. After 3 weeks treatment, mice were sacrificed and primary tumors and lung metastases were evaluated with fluorescence imaging. AIIB2 significantly inhibited spontaneous lung metastasis but not primary tumor growth, possibly due to inhibition of lung seeding of the cancer cells as imaged in the experimental metastasis study. AIIB2 treatment also increased survival of mice with orthotopically-growing 143B-RFP. © 2012 Wiley-Liss, Inc.

Stability of many tumor-relevant proteins is partly mediated by E3 ligases which determine substrate-specificity within the ubiquitin system. Recent data demonstrated that increased nuclear expression of the E3 ligase seven in absentia homologue (SIAH)-1 in human hepatocarcinogenesis supports tumor cell proliferation and migration. To define whether closely related SIAH-2 synergizes with pro-tumorigenic SIAH-1, we systematically analysed expression, localization, and functional relevance of SIAH-2 in human hepatocellular carcinoma (HCC). Nuclear accumulation of SIAH-2 is detectable in more than 60% of all HCCs and correlates with tumor progression, cell proliferation, and distant metastasis. An inverse correlation between nuclear SIAH-1 and SIAH-2 was detected, suggesting independent mechanisms for nuclear enrichment. Inhibition of nuclear SIAH-2 by RNAi in HCC cell lines reduced proliferation as well as lateral tumor cell motility and transmigration; however, combined knock down of both, SIAH-1 and SIAH-2 did not further amplify biological effects compared to single gene inhibition. Reduction of SIAH-2 expression sensitizes HCC cells to the treatment with different cytostatic drugs, demonstrating that SIAH-2-targeting approaches may increase the response of HCC cells to conventional chemotherapy. Together, these data show that SIAH-2 - as described for SIAH-1 - accumulates in nuclei of HCC cells where it supports tumor growth and tumor cell dissemination. Because the nuclear pattern of SIAH-2 differs in HCC tissues from the SIAH-1 pattern and because the inactivation of SIAH-2 is not compensated by SIAH-1, the specific inhibition of SIAH-2 (especially in combination with other drugs) represents a promising therapeutic strategy for HCC. © 2012 Wiley-Liss, Inc.

Ewing's sarcoma family of tumors (EFT) are characterized by the presence of chromosomal translocations leading to expression of oncogenic transcription factors such as, in the majority of cases, EWS/FLI1. Due to its key role in Ewing's sarcoma development and maintenance, EWS/FLI1 represents an attractive therapeutic target. Here, we characterize PHLDA1 as a novel direct target gene whose expression is repressed by EWS/FLI1. Using this gene and additional specific well-characterized target genes such as NROB1, NKX2.5, and CAV1, all activated by EWS/FLI1, as a read-out system, we screened a small-molecule compound library enriched for FDA-approved drugs that modulated the expression of EWS/FLI1 target genes. Among a hit-list of 9 well known drugs such as camptothecin, fenretinide, etoposide and doxorubicin, we also identified the kinase inhibitor midostaurin (PKC412). Subsequent experiments demonstrated that midostaurin is able to induce apoptosis in a panel of 6 Ewing's sarcoma cell lines in vitro and can significantly suppress xenograft tumor growth in vivo.

These results suggest that midostaurin might be a novel drug that is active against Ewing's cells which might act by modulating the expression of EWS/FLI1 target genes. © 2012 Wiley-Liss, Inc.

Genotyping may improve risk stratification of high-risk (HR) HPV-positive women in cervical screening programs, but prospective data comparing the natural history and carcinogenic potential of individual HR types remain limited. A meta-analysis of cross-sectional HR HPV type distribution in 115,789 HPV-positive women was performed, including 33,154 normal cytology, 6,810 atypical squamous cells of undetermined significance (ASCUS), 13,480 low-grade squamous intraepithelial lesions (LSIL) and 6,616 high-grade SIL (HSIL) diagnosed cytologically, 8,106 cervical intraepithelial neoplasia grade 1 (CIN1), 4,068 CIN2 and 10,753 CIN3 diagnosed histologically, and 36,374 invasive cervical cancers (ICC), from 423 PCR-based studies worldwide. No strong differences in HPV type distribution were apparent between normal cytology, ASCUS, LSIL or CIN1. However, HPV16-positivity increased steeply from normal/ASCUS/LSIL/CIN1 (20-28%), through CIN2/HSIL (40/47%) to CIN3/ICC (58/63%). HPV16, 18 and 45 accounted for a greater or equal proportion of HPV infections in ICC compared to normal cytology (ICC:normal ratios=3.07, 1.87 and 1.10, respectively) and to CIN3 (ICC:CIN3 ratios=1.08, 2.11 and 1.47, respectively). Other HR types accounted for important proportions of HPV positive CIN2 and CIN3, but their contribution dropped in ICC, with ICC:normal ratios ranging from 0.94 for HPV33 down to 0.16 for HPV51. ICC:normal ratios were particularly high for HPV45 in Africa (1.85) and South/Central America (1.79), and for HPV58 in Eastern Asia (1.36). ASCUS and LSIL appear proxies of HPV infection rather than cancer precursors, and even CIN3 is not entirely representative of the types causing ICC. HPV16 in particular, but also HPV18 and 45, warrant special attention in HPV-based screening programs. © 2012 Wiley-Liss, Inc.

Disulfiram (DSF) is an aldehyde dehydrogenase inhibitor currently used for the treatment of alcoholism.

Here we show that multiple myeloma (MM) cell lines and primary cells from newly diagnosed and relapsed/resistant patients affected by MM, acute myeloid and lymphoblastic leukaemia (AML and ALL) are significantly sensitive to DSF alone and in combination with copper.

These effects are present at doses lower than those achievable in vivo after DSF standard administration. The cytotoxic effect achieved by this treatment is comparable to that obtained by conventional chemotherapy and is absent in normal hematopoietic cells. In addition, we found that DSF plus copper induces loss of mithocondrial membrane potential, triggers reactive oxygen species (ROS) production and activate executioner caspases. DSF-copper-induced apoptosis and caspases activation is strongly reversed by antioxidant N-acetylcysteine, thus indicating a critical role of ROS. These results might suggest the use of the old drug DSF, alone or in combination with copper, in the treatment of haematological malignancies.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus (HTLV-1). SIRT1, a nicotinamide adenine dinucleotide (NAD⁺)-dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, owing to its ability to deacetylate numerous substrates, such as histone and NF-κB, which is implicated as an exacerbation factor in ATL. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV-1-related cell lines. Sirtinol-induced apoptosis was mediated by activation of the caspase family, and degradation of SIRT1 in the nucleus. Furthermore, SIRT1-knockdown by SIRT1-specific small interfering RNA caused apoptosis via activation of caspase-3 and PARP in MT-2 cells, HTLV-1-related cell line. These results suggest that SIRT1 is a crucial anti-apoptotic molecule in ATL cells, and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL.

Thyroid hormones influence both normal breast cell differentiation and breast cancer cell proliferation and stimulate the angiogenesis of certain cancer forms. Several cross-sectional studies have measured thyroid hormones / auto antibodies in breast cancer ceases vs. controls, but it is difficult to determine the cause-effect direction in these studies. Only three prospective studies have reported on the subject so far. The aim of the present study was to investigate pre-diagnostically measured levels of thyroid hormones, thyrotropin, and thyroid autoantibodies in relation to subsequent risk of breast cancer.

The Malmoe Diet and Cancer study examined 17,035 women between 1991 and 1996. Blood samples were collected at baseline and free T3, free T4, TSH, and TPO-Ab levels were measured in 676 cases and 680 controls. Relative risks with 95% confidence intervals were assessed using a logistic regression analysis adjusted for potential confounders.

Free T4 levels were positively associated with a high risk of breast cancer, and the OR for women with free T4 levels above vs. below the median was 1.40 (1.10-1.77). This association was most pronounced in overweight women (1.51:1.07-2.12). Women with high levels of TPO-Ab had a lower risk of breast cancer, but only the analysis of TPO-Ab as a continuous variable reached statistical significance.

Free T4 was in this study positively associated with a high risk of breast cancer. This association was most pronounced in overweight/obese women. Women with a high level of TPO-Ab had a relatively low risk of breast cancer. © 2012 Wiley-Liss, Inc.

Preclinical studies and clinical analyses have implicated the mammalian target of rapamycin (mTOR) pathway in the progression of prostate cancer, suggesting mTOR as a potential target for new therapies. mTOR, a serine/threonine kinase, belongs to two distinct signaling complexes: mTORC1 and mTORC2. We previously showed that the synthetic organoselenium compound, p-XSC, effectively inhibits viability and critical signaling molecules (e.g., androgen receptor, Akt) in androgen responsive (AR) and androgen independent (AI) human prostate cancer cells. Based on its inhibition of Akt, we hypothesized that p-XSC modulates mTORC2, an upstream regulator of the kinase. We further hypothesized that combining p-XSC with rapamycin, an mTORC1 inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer. The effects of p-XSC and rapamycin, alone or in combination, on viability and mTOR signaling were examined in AR LNCaP prostate cancer cells and AI C4-2 and DU145 cells. Phosphorylation of downstream targets of mTORC1 and mTORC2 was analyzed by immunoblotting. The interaction of mTORC1- and mTORC2-specific proteins with mTOR was probed through immunoprecipitation and immunoblotting. p-XSC inhibited phosphorylation of mTORC2 downstream targets, Akt and PCKa, and decreased the levels of rictor, an mTORC2-specific protein, co-immunoprecipitated with mTOR in C4-2 cells. The combination of p-XSC and rapamycin more effectively inhibited viability and mTOR signaling in C4-2, LNCaP, and DU145 cells than either agent individually. © 2012 Wiley-Liss, Inc.

Expression of vimentin and the epithelial to mesenchymal transition (EMT) markers E-cadherin, β-catenin is essential for the progression of various human cancers. This study aimed to investigate the aberrant localization E-cadherin, β-catenin and vimentin, and their prognostic significance in 122 NPC patients by immunohistochemistry and immunofluorescence. Our results showed that both membranous and cytoplasmic localization of E-cadherin staining were associated with lymph node metastasis (p = 0.000 and 0.005, respectively) and clinical stage (p = 0.000 and 0.007, respectively). High cytoplasmic β-catenin correlated significantly with larger tumor size (p = 0.020), lymph node metastasis (p = 0.000) and advanced clinical stage (p = 0.036). However, no significant difference was observed between membranous β-catenin and clinicopathologic features (p = 0.05). High nuclear vimentin expression correlated significantly with positive lymph node metastasis (p = 0.000) and advanced clinical stage (p = 0.000). Multivariate analysis showed that nuclear vimentin and cytoplasmic E-cadherin were independent prognostic factors (p = 0.016 and 0.001, respectively), as well as M classification (p = 0.001). More importantly, patients with high coexpression of nuclear vimentin and cytoplasmic E-cadherin had shorter survival time (p = 0.000). Furthermore, high coexpression of these two proteins was closely associated with lymph node metastasis (p = 0.000) and advanced clinical stage (p = 0.000). Our studies provide convincing evidence that EMT may play an important role in the biological progression of NPC, and nuclear vimentin and cytoplasmic E-cadherin might have independent prognostic value in NPC patients and serve as novel targets for prognostic therapeutics. © 2012 Wiley-Liss, Inc.

The studies reporting population-based estimates of the proportion of children with a parent suffering from cancer are very few. These children have been shown to suffer from psychological symptoms, but it is not known whether their use of psychiatric services is increased. This study examined the prevalence of children affected by parental cancer at national level and whether these children use specialised psychiatric services more than their peers. The study is a retrospective population-based registry study. All 60,069 children born in Finland in 1987 were followed up with various health and social registers from 1987 to 2008. The associations of parental cancer treatments with children's psychiatric service use were analysed with logistic regressions. During the 21-year follow-up 3,909 (6.6%) of the children had a parent suffering from cancer. The children of the cancer patients used more specialised psychiatric care than their peers and the service use depended on parent's gender, as well as cohort members' gender and the age at occurrence. The combination of parental cancer and psychiatric disorder, whether the ill parent or spouse, increased the children's psychiatric service use even more. Children affected by parental cancer comprise a substantial part of the population in society using increased level of psychiatric services. Parental cancer is clearly an illness which has to be taken into account in planning child- and parentingfocused prevention and promotion actions in adult health care. © 2012 Wiley-Liss, Inc.

Thymidylate synthase (TS) is an important enzyme involved in folate metabolism and catalyzes methylation of dUMP to dTMP, which is essential for DNA replication. TSER and TS1494del6, two functionally important and ethnically diverse polymorphisms mapping to its gene region, are the most extensively studied. Considering the potential influence of altering TS activity, it is plausible that TS polymorphisms might play a role in the development of cancer. Although the effects of TS polymorphisms on susceptibility to human cancer have been investigated in many studies, the results remain conflicting rather than conclusive. In order to resolve these conflicts, we performed a quantitative synthesis of the evidence on the association between these two polymorphisms and cancer risk, including 63 studies (19,707 cases and 27,398 controls) for TSER polymorphism and 39 studies (13,489 cases and 16,297 controls) for TS1494del6 polymorphism. Our meta-analysis suggested that these two polymorphisms are not associated with cancer risk when all studies were pooled together. In the stratified analyses, we found that individuals with 2R/2R genotype had a significantly higher cancer risks among Asians (2R/2R vs. 3R/3R: OR=1.24, 95% CI=1.05–1.45; recessive model: OR=1.23, 95%CI=1.05–1.44). Further analyses revealed that 2R/2R genotype was significantly associated with an increased risk of gastroesophageal cancer among Asians, whereas it might provide protecting effects against colorectal cancer risk in a dominant genetic model for Caucasians. Additionally, TS1494del6 polymorphism may contribute to genetic susceptibility of breast cancer among Asians. © 2012 Wiley-Liss, Inc.

Several industrialized nations recommend fecal occult blood testing (FOBT) to screen for colorectal cancer (CRC), but corresponding screening guidelines do not specify how individuals with a prior false positive FOBT result (fpFOBT) should be managed in terms of subsequent CRC screening. Accordingly, we conducted a decision analysis to compare different strategies for managing such individuals.

We used a previously-developed CRC microsimulation model, SimCRC, to calculate life-years and the lifetime number of colonoscopies (as a measure of required resources) for a cohort of 50-year-olds to whom FOBT-based CRC screening is offered annually from age 50 to 75. We compared three management strategies for individuals with a prior fpFOBT: 1) resume screening in 10 years with 10-yearly colonoscopy (SwitchCol_long); 2) resume screening in 1 year with annual FOBT (ContinueFOBT_Short); and 3) resume screening in 10 years (i.e., the recommended interval following a negative colonscopy) with annual FOBT (ContinueFOBT_long). We performed sensitivity analyses on various parameters and assumptions.

When using different management strategies for individuals with a prior fpFOBT the variation in the number of life-years gained relative to no screening was less than 2%, while the variation in the lifetime number of colonoscopies was 23% (percentages are calculated as the maximum difference across strategies divided by the lowest number across strategies). The ContinueFOBT_long strategy showed the lowest lifetime number of colonoscopies per life-year gained even when key assumptions were varied.

In conclusion, the ContinueFOBT_long strategy was advantageous regarding both clinical benefit and required resources. Specifying an appropriate management strategy for individuals with a prior fpFOBT may substantially reduce required resources within a FOBT-based CRC screening program without limiting its effectiveness. © 2012 Wiley-Liss, Inc.

Registry based cancer incidence and mortality data are widely used for etiologic research, cancer control and health care monitoring and planning. The complete coverage of all cases is the key criteria of data quality but it is difficult to assess because the alternative sources of data may be flawed. The aim of this study was to examine, at a nationwide level, the completeness of the Swedish Cancer Registry (CR) regarding persons who died of cancer, based on the Cause of Death Registry (DR), and using the Hospital Discharge Registry (HDR) as an additional source of data. Individuals who died of cancer from years 1999 through 2008 recorded in DR were linked to CR and HDR. A total of 190,692 individuals were identified from DR with cancer as the underlying cause of death; the mean identification rate of concordant cancer in CR was 79.8%, depending on tumor site and age at death. Breast, bladder and prostate cancers showed the highest rate of identification whereas bone, liver and pancreatic cancers showed the lowest rate of identification. CR had no records on 10.6% of cancer cases recorded in DR. Similarly, the identification rate in HDR was 84.5% for concordant cancer and with 9.6% of cases missing. Neither source reported cancers for 3.4% of cancer cases recorded in DR. In conclusion, some 10% of cancer deaths had no cancer records in CR or in HDR, and 3.4% were missing in both sources. The identification rate depended on tumor site, age at death, and, to some extent, death outside hospital. © 2012 Wiley-Liss, Inc.

The prognostic impact of distinct KRAS mutations in colorectal carcinomas is not fully characterized. We hypothesized that the prognostic impact of KRAS mutations is modulated by KRAS mutant allele specific imbalance (MASI). KRAS MASI was assessed by sequencing electropherograms in KRAS-mutated colorectal carcinomas (N = 394, prospectively tested). The mechanism of KRAS MASI was studied by fluorescence in situ hybridization (FISH) (N = 50). FISH showed that KRAS MASI developed by chromosome 12 hyperploidy (9/18, 50%) or KRAS amplification (1/18, 5.5%). KRAS MASI was more common in tumors with KRAS codon 13 than with codon 12 mutations (24/81, 30% vs. 54/313, 17%; odds ratio [OR], 2.0, 95% confidence interval [CI], 1.2 – 3.5; P = 0.01). KRAS MASI was correlated with overall survival (N = 358, median follow-up – 21 months). In a multivariate analysis, KRAS codon 13 MASI was an independent adverse prognostic factor (compared to codon 13 mutants without MASI combined with all codon 12 mutants) (adjusted hazard ratio, 2.2, 95% CI 1.2 – 3.9; P = 0.01).

KRAS MASI arises through chromosome 12 hyperploidy or KRAS amplification, and, when affects KRAS codon 13, is associated with worse overall survival. © 2012 Wiley-Liss, Inc.

The recently described combined carbogen USPIO (CUSPIO) magnetic resonance imaging (MRI) method uses spatial correlations in independent imaging biomarkers to assess specific components of tumour vascular structure and function. This study aimed to evaluate CUSPIO biomarkers for the assessment of tumour response to anti-angiogenic therapy. CUSPIO imaging was performed in subcutaneous rat C6 gliomas before and 2 days after treatment with the potent VEGF signalling inhibitor cediranib (n=12), or vehicle (n=12). Histological validation of Hoechst 33342 uptake (perfusion), smooth muscle actin staining (maturation), pimonidazole adduct formation (hypoxia) and necrosis were sought. Following treatment, there was a significant decrease in fractional blood volume (-43%, p<0.01) and a significant increase in haemodynamic vascular functionality (treatment altered ΔR₂^*_carbogen from 1.2 to -0.2 s^-1, p<0.05). CUSPIO imaging revealed an overall significant decrease in plasma perfusion (-27%, p<0.05) following cediranib treatment, that was associated with selective effects on immature blood vessels. The CUSPIO responses were associated with a significant 15% reduction in Hoechst 33342 uptake (p<0.05), but no significant difference in vascular maturation or necrosis. Additionally, treatment with cediranib resulted in a significant 40% increase in tumour hypoxia (p<0.05). The CUSPIO imaging method provides novel and more specific biomarkers of tumour vessel maturity and vascular haemodynamics, and their response to VEGF signalling inhibition, compared to current MR imaging biomarkers utilised in the clinic. Such biomarkers may prove effective in longitudinally monitoring tumour vascular remodelling and/or evasive resistance in response to anti-angiogenic therapy. © 2012 Wiley-Liss, Inc.

Dichlorodiphenyltrichloroethane (p,p'-DDT), an organochlorine pesticide known to have deleterious health effects in humans, has been linked to hepatocellular carcinoma (HCC) in rodents. A recent study has reported that p,p'-DDT and its most persistent metabolite, dichlorodiphenyldichloroethylene (p,p'-DDE), may also be associated with HCC in humans. To examine whether there is an association between p,p'-DDT and/or p,p'-DDE in a population at high-risk of developing HCC.

A nested case-control study was conducted within the 83,794 person Haimen City Cohort in China. Sera and questionnaire data were collected from all participants between 1992 and 1993. The current study included 473 persons who developed HCC and 492 who did not, frequency matched on sex, age and area of residence. p,p'-DDT and p,p'-DDE levels were determined by mass spectrometry. Hepatitis B viral infection status (based on hepatitis B virus surface antigen; HBsAg) was also determined. Adjusting for age, sex, area of residence, HBsAg, family history of HCC, history of acute hepatitis, smoking, alcohol, occupation (farmer vs. other) and levels of p,p'-DDT or p,p'-DDE, odds ratios (OR) and 95% confidence intervals (CI) were calculated via unconditional logistic regression, p,p'-DDT and/or p,p'-DDE serum levels were significantly associated with sex, area of residence, occupation, alcohol consumption and cigarette smoking. Overall, the highest quintile of p,p'-DDT was associated with an increased risk of HCC, OR = 2.96 95% CI; 1.19-7.40. There were no statistically significant associations with p,p'-DDE. Overall, these results suggest that recent exposure to p,p'-DDT may increase risk of HCC. © 2012 Wiley-Liss, Inc.

Mesenchymal stem cells (MSCs) are non haematopoietic multipotent adult stem cells. They have been shown to have a natural tropism for many tumours types, including colorectal, and are capable of escaping host immune surveillance. MSCs are known to engraft at tumours and integrate into their architecture, potentially as carcinoma associated fibroblasts (CAFs). In contrast with other malignancies, our understanding of the interactions between colorectal cancer cells and MSCs remains limited. Considering the established importance of inflammation in the colorectal cancer primary tumour microenvironment and the role of stromal cells in this process, there is a potential wealth of information to be gleaned from further investigation of interactions between these cell populations. Epithelial-mesenchymal transition is central to colorectal cancer progression and MSCs have also been implicated in this process. This review explores the current knowledge (both in vitro and in vivo) of interactions between colorectal cancer cells and MSCs. It highlights potential effects of cell source, number and ratio on outcome of in vivo studies, and explores strategies to more accurately explore their role in the primary tumour microenvironment. As our understanding of the underlying molecular processes in colorectal cancer develops, elucidation of these interactions will be central to development of novel therapeutic strategies for this prevalent disease. © 2012 Wiley-Liss, Inc.

Obesity strongly increases risk of endometrial cancer and is projected to increase current and future endometrial cancer incidence. In order to fully understand endometrial cancer incidence, one should also examine both hysterectomy, which eliminates future risk of endometrial cancer, and endometrial hyperplasia (EH), a precursor that prompts treatment (including hysterectomy). Hysterectomy and EH are more common than endometrial cancer, but data on simultaneous temporal trends of EH, hysterectomy, and endometrial cancer are lacking. We used linked pathology, tumor registry, surgery, and administrative datasets at the Kaiser Permanente Northwest Health Plan to calculate age-adjusted and age-specific rates, 1980-2003, of EH only (N=5990), EH plus hysterectomy (N=904), hysterectomy without a diagnosis of EH or cancer (N=14,926), and endometrial cancer (N=1208). Joinpoint regression identified inflection points and quantified annual percentage changes (APCs). The EH APCs were -5.3% (95% confidence interval [CI]= -7.4% to -3.2%) for 1980-1990, -12.9% (95% CI = -15.6% to -10.1%) for 1990-1999; and 2.4% (95% CI = -6.6% to 12.2%) for 1999-2003. The EH-plus-hysterectomy APCs were -8.6% (95% CI = -10.6% to -6.5%) for 1980-2000 and 24.5% (95% CI = -16.5% to 85.7%) for 2000-2003. Hysterectomy rates did not significantly change over time. The endometrial cancer APCs were -6.5% (95% CI = -10.3% to -2.6%) for 1980-1988 and 1.4% (95% CI = -0.2% to 3.0%) for 1988-2003. Hysterectomy rates were unchanged, but increased endometrial cancer incidence after 1988 and the reversal, in 1999, of the longstanding decline in EH incidence could reflect the influence of obesity on endometrial neoplasia. © 2012 Wiley-Liss, Inc.

Pancreatic cancer is the one of most common causes of cancer deaths and has the worst prognosis. Clinical observational studies suggest that statins may reduce the risk of pancreatic cancer. The chemopreventive efficacy of the statin atorvastatin (Lipitor®) and the role of the phosphatidyl-inositol 3-kinase(PI3/AKT) signaling pathway were evaluated for the progression of pancreatic intraepithelial neoplasms (PanINs) to pancreatic ductal adenocarcinoma (PDAC) in conditional p48^Cre/+-LSL-Kras^G12D/+ transgenic mice. Six-week old male p48^Cre/+-LSL-Kras^G12D/+ (20/group) mice were fed AIN-76A diets containing 0, 200, and 400 ppm atorvastatin for 35 weeks. At termination, pancreata were evaluated histopathologically for PanINs and PDAC, and for various PI3/AKT signaling markers, and inflammatory cytokines, by immunohistochemistry/ immunohistoflourscence, ELISA, Western blotting and/or Reverse Transcription-PCR methods. Control diet-fed mice showed 85% incidence of PDAC; whereas, mice fed with atorvastatin showed PDAC incidence of 65 and 35% respectively (p<0.0001). Similarly, significant suppression of PanIN-3 (22.6%) was observed in mice fed 400 ppm atorvastatin. Importantly, pancreata from atorvastatin-treated mice were 68% free from ductal lesions. Furthermore, pancreas of mice administered with atorvastatin had significantly reduced expressions levels of PCNA, p2X7, p-ERK, RhoA, cyclin D1, survivin, Akt, pAKT, β-catenin, cyclin E, cdK2, and caveolin-1. Also, atorvastatin-treated mice had shown dose-dependent suppression of inflammatory cytokines and a significant increase in tunnel-positive cells, p21 and PARP expression levels in pancreas. Atorvastatin significantly delays the progression of PanIN-1 and -2 lesions to PanIN-3 and PDAC by modulating PI3/AKT signal molecules in a preclinical model, suggesting potential clinical benefits of statins for high risk pancreatic cancer patients. © 2012 Wiley-Liss, Inc.

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 that catalyzes the trimethylation of histone H3 on Lys 27, and represses gene transcription. EZH2 enhances cancer-cell proliferation and regulates stem cell maintenance and differentiation. Here, we demonstrate that EZH2 is highly expressed in medulloblastoma, a highly malignant brain tumor of childhood, and this altered expression is correlated with genomic gain of chromosome 7 in a subset of medulloblastoma. Inhibition of EZH2 by RNAi suppresses medulloblastoma tumor cell growth. We show that 3-deazaneplanocin A, a chemical inhibitor of EZH2, can suppress medulloblastoma cell growth partially by inducing apoptosis. Suppression of EZH2 expression diminishes the ability of tumor cells to form spheres in culture and strongly represses the ability of known oncogenes to transform neural stem cells. These findings establish a role of EZH2 in medulloblastoma and identify EZH2 as a potential therapeutic target especially in high-risk tumors. © 2012 Wiley-Liss, Inc.

Molecular characterization has been extensively studied in serrated polyps but very little is known in serrated adenocarcinomas (SACs). We analyzed the incidence of KRAS, BRAF and PIK3CA mutations, microsatellite instability (MSI) status and loss of the DNA repair proteins MLH1, MSH2, MSH6 and MGMT in a series of 89 SAC, 81 matched conventional carcinomas (CC) and 13 sporadic colorectal cancer showing histological and molecular features of high-level MSI (sMSI-H). Our results demonstrate that KRAS are more prevalent than BRAF mutations in SAC (42.7% vs. 25.8%;p=0.011) being the KRAS-mutated cases even more abundant in SAC displaying adjacent serrated adenomas (51%). G12D and E545K are the most common KRAS and PIK3CA mutations found in SAC respectively. SAC show higher frequency of MGMT loss compared to CC (50.6% vs. 25.3%;p=0.001) especially in distal colon/rectum (60.0% vs. 21.6%;p=0.0009). SAC differ from sMSI-H in terms of KRAS and BRAF mutation prevalence, MSI status and MLH1 expression (p=0.0003, p>0.0001, p>0.0001, p>0.001, respectively). SACs are more often KRAS-mutated and microsatellite stable and display different molecular and immunohistochemical characteristics compared to CC and sMSI-H. © 2012 Wiley-Liss, Inc.

Cysteine cathepsins play an important role in shaping the highly infiltrative growth pattern of human gliomas. We have previously demonstrated that the activity of cysteine cathepsins is elevated in invasive GBM cells in vitro, in part due to attenuation of their endogenous inhibitors, the cystatins. To investigate this relationship in vivo, we established U87-MG xenografts in NOD/SCID-eGFP mice. Here, tumour growth correlated with an elevated enzymatic activity of CatB both in the tumour core and at the periphery, whereas CatS and CatL levels were higher at the xenograft edge compared to the core. Reversely, StefB expression was detected in the tumour core, but it was generally absent in the tumour periphery, suggesting that down-regulation of this inhibitor correlates with in vivo invasion. In human GBM samples, all cathepsins were elevated at the tumour periphery compared to brain parenchyma. CatB was also typically associated with angiogenic endothelia and necrotic areas. StefB was mainly detected in the tumour core, whereas CysC and StefA were evenly distributed, reflecting the observations in the xenografts. However, at the mRNA level, no differences in cathepsins and cystatins were observed between the tumour centre and the periphery in both human biopsies and xenografts. Interestingly, in human tumours, cathepsin and stefin transcript levels correlated with CD68 and CXCR4 levels, but not with EGFR. Moreover, we reveal for the first time that an elevated StefA mRNA level is a highly significant prognostic factor for patient survival. © 2012 Wiley-Liss, Inc.

The purpose of this study was to use a near-infrared (NIR) fluorescent cyclic His-Try-Gly-Phe peptide to characterize and image the expressions of matrix metalloproteinases (MMPs), which are correlated with cancer promotion, in an inflammation-induced colorectal cancer (ICRC) model. We explored the relationship between the development of colon cancer and the expression of MMPs at the same colonic sites in ICRC models. To develop ICRC models, mice were administered a single intraperitoneal dose (10 mg/kg) of azoxymethane (AOM) and exposed orally to 2% dextran sodium sulfate (DSS) for one week. MMP-2 expression and β-catenin activation in colonic lesions were characterized by immunohistochemical (IHC) staining. After being treated with inducers for some time, cancerous lesions were found to express high β-catenin and MMP-2. The profiles of MMP expression were correlated with β-catenin activation in the colonic lesions. c(KAHWGFTLD)NH₂ (C6) peptide was prepared by standard Fmoc peptide synthesis to target MMPs. Molecular weight of Cy5.5-C6 was 1954.78 g/mol (calculated MW = 1955.23 g/mol). The in vitro characterization of Cy5.5-C6 showed MMP binding specificity in a cell experiment. In vivo NIRF imaging showed high accumulation of Cy5.5-C6 in tumors with associated expression of MMP-2 in colonic lesions after intravenous injection. The MMP-2 specificity of Cy5.5-C6 was confirmed by successful inhibition of probe uptake in the tumor due to the presence of excess C6 peptide. The use of Cy5.5-C6 to target MMP-2 has the potential to be developed into an effective molecular imaging agent to monitor ICRC progress. © 2012 Wiley-Liss, Inc.

Neuroblastoma (NB) is a frequently lethal tumour that occurs in childhood and originates from embryonic neural crest cells. The malignant and aggressive phenotype of NB is strictly related to the de-regulation of pivotal pathways governing the proliferation/differentiation status of neural crest precursor cells, such as MYCN, Delta/Notch, and Wnt/β-catenin (CTNNB1) signalling. In this article, we demonstrate that sialidase NEU4L influences the differentiation/proliferation behaviour of NB SK-N-BE cells by determining hyperactivation of the Wnt/ß-catenin signalling pathway. NEU4L overexpression in SK-N-BE cells induced significant increases in active, non-phosphorylated β-catenin content, β-catenin/TCF transcriptional activity, and β-catenin gene target expression including MYCN, MYC, CCND2 (cyclin D2), and CDC25A. In turn, these molecular features strongly modified the behaviour of NEU4L SK-N-BE overexpressing cells, promoting the following: a) an enhanced proliferation rate, mainly due to a faster transition from G1 to S phase in the cell cycle; b) a more undifferentiated cell phenotype, which was similar to stem-like NB cells and possibly mediated by an increase of the expression of the pluripotency genes, MYC, NANOG, OCT-4, CD133, and NES (nestin); and c) the failure of NB cell differentiation after serum withdrawal. The molecular link between NEU4L and Wnt/β-catenin signalling appeared to rely most likely on the capability of the enzyme to modify the sialylation level of cell glycoproteins. These findings could provide a new candidate for therapeutic treatment. © 2012 Wiley-Liss, Inc.

Tumor cells can escape from cytotoxic T-cell responses by downregulation of human leukocyte antigen (HLA) class I molecules expressed at the cell surface which has been associated with a deficient mismatch repair (MMR) system in colorectal carcinomas. This study investigated the association between expression of MMR proteins and HLA class I in sporadic endometrioid endometrial carcinomas (EC). In a consecutively selected cohort of 486 EC patients, MMR proteins (MLH1, MSH2 and MSH6) and HLA class I (HLA-A, -B, -C or β₂m) were investigated by immunohistochemistry. Expression levels of MMR proteins and HLA class I were compared between low-grade and high-grade EC's. HLA class I expression was compared between tumors with loss (negative immunostaining of ?1 MMR protein) and expression of MMR proteins. Associations between previously determined numbers of intratumoral CD8⁺ T-lymphocytes and expression of MMR proteins and HLA class I and the influence on survival was determined. EC's with loss of MMR protein expression (33.5%) more frequently have loss of HLA-B/C (37.3%), compared to EC's with MMR protein expression (25.5%, p=0.007). Patients with loss of MMR proteins have a worse disease-specific survival compared to patients with expression (p=0.039). CD8⁺ T-lymphocytes have a positive influence on disease-free and disease-specific survival in the total EC cohort but not in patients with loss of MMR protein expression. In conclusion, our results indicate that loss of MMR protein expression is related to selective downregulation of HLA class I which contributes to immune escape in EC with an abnormal MMR system. © 2012 Wiley-Liss, Inc.

Hypoxia is known to play important roles in the development and progression of tumors. We previously demonstrated that S100A4, a critical molecule for metastasis, was upregulated in ovarian cancer cells. Therefore, we examined the mechanisms of the upregulation of S100A4 expression in ovarian carcinoma cells, with particular attention paid to the effects of hypoxia. The expression levels of S100A4 were found to be correlated with the invasiveness of ovarian carcinoma cells in vitro and in vivo, and the upregulation of S100A4 expression was associated with hypomethylation of CpG sites in the 1^st intron of S100A4 in ovarian carcinoma cell lines and tissues. The expression of S100A4 was increased under hypoxia and was associated with elevated invasiveness, which was inhibited by S100A4 siRNA. In addition, exposure to hypoxia reduced the methylation of hypoxia-response elements (HRE) of the S100A4 gene in a time-dependent fashion, in association with the increased binding of HIF-1α to a methylation-free HRE in ovarian carcinoma cells. These results indicate that hypoxia-induced hypomethylation plays an essential role in S100A4 overexpression and the epigenetic transformation of ovarian carcinoma cells into the “metastatic phenotype”. © 2012 Wiley-Liss, Inc.

Previous studies report an atypical cancer pattern among patients with Parkinson disease. Here we evaluate the cancer pattern among people diagnosed with Parkinson disease in an extension of our previous cohort study. For this Danish population based cohort study we identified 20,000 people with Parkinson disease diagnosed in 1977–2006, from the National Danish Hospital Register. Cohort members were followed up for cancer in the Danish Cancer Registry until 31 December 2008, and their incidence rates of cancer were compared with age-, sex- and calendar period-specific rates in the general population as standardized incidence rate ratios (SIRs). In sub analyses we estimated the risk for cancer among patients with early-onset Parkinson disease and also, we compared breast tumor characteristics among Parkinson disease women to that of a control group. The overall cancer risk in our cohort was decreased (SIR, 0.86; 95% confidence interval (CI), 0.83–0.90), as were those for smoking-related (SIR, 0.65; 95% CI, 0.60–0.70) and non-smoking related cancers (SIR, 0.79; 95% CI, 0.71–0.86). The cohort had increased risks for malignant melanoma (SIR, 1.41; 95% CI, 1.09–1.80), non-melanoma skin cancer (1.29; 1.18–1.39) and female breast cancer (1.17; 1.02–1.34). Among patients with early-onset Parkinson disease the risk for cancer was comparable to that of the general population. Of breast tumor characteristics only grade of malignancy differed between Parkinson disease women and controls. This study confirms a lower cancer risk among people with Parkinson disease. Increased risks for malignant melanoma, non-melanoma skin cancer and breast cancer might be due to shared risk factors with Parkinson disease.

The RON receptor tyrosine kinase is overexpressed and stimulates invasive growth in pancreatic cancer cells, yet the mechanisms that underlie RON-mediated phenotypes remain poorly characterized. To better understand RON function in pancreatic cancer cells, we sought to identify novel RON interactants using multidimensional protein identification analysis. These studies revealed plectin, a large protein of the spectrin superfamily, to be a novel RON interactant. Plectin is a multifunctional protein that complexes with integrin-β4 (ITGB4) to form hemidesmosomes, serves as a scaffolding platform crucial to the function of numerous protein signaling pathways, and was recently described as an overexpressed protein in pancreatic cancer ^1-2. In this study we demonstrate that upon exposure to its ligand, macrophage stimulating protein, RON binds to plectin and ITGB4, which results in disruption of the plectin-ITGB4 interaction and enhanced cell migration, a phenotype that can be recapitulated by shRNA mediated suppression of plectin expression. We demonstrate that disruption of plectin-ITGB4 is dependent on RON and PI3 kinase, but not MEK, activity. Thus, in pancreatic cancer cells, plectin and ITGB4 form hemidesmosomes which serve to anchor cells to the ECM and restrain migration. The current study defines a novel interaction between RON and plectin, provides new insight into RON mediated migration and further supports efforts to target RON kinase activity in pancreatic cancer.

Impairment of endogenous differentiation pathways like retinoic acid (RA) signaling seems to be a central pathogenetic event in astrocytic gliomas. Among others, expression of the differentiation-promoting RA chaperon protein cellular retinoic acid binding protein 2 (CRABP2) is extenuated in high-grade gliomas. Against this background, we aimed at identifying potential pathomechanisms underlying reduced CRABP2 expression in these tumors. Employing MassARRAY methylation analysis we detected extensive CpG methylation upstream of the CRABP2 gene locus in a study sample comprising 100 astrocytic gliomas of WHO grade II to IV. Compared to non-tumorous control samples tumors revealed increased CpG methylation and methylation levels were inversely correlated to CRABP2 mRNA expression. Substantiating our in situ findings, CRABP2 mRNA levels increased in glioma cell lines after exposure to the demethylating agent 5-aza-2'-deoxycytidine. Finally, a distinct CpG methylation signature distinguished between primary glioblastoma on the one hand and the group of astrocytoma WHO II-III and secondary glioblastoma on the other hand. Altogether, our observations suggest that epigenetic silencing of CRABP2 might contribute to an immature phenotype in glioma cells.

Results of studies that examined the relationship between physical activity and non-Hodgkin lymphoid neoplasms (NHL) are inconsistent, and only one study to date examined time spent sitting in relation to NHL. We examined recreational physical activity and leisure-time sitting in relation to risk of NHL in the American Cancer Society Cancer Prevention Study-II Nutrition Cohort. Between 1992 and 2007, 2,002 incident cases were identified among 146,850 participants who were cancer-free at enrollment. Cox proportional hazards regression was used to compute hazard ratios (HR) and 95% confidence intervals (CI) while adjusting for potential confounders. Women who sat for at least six hours/day were at 28% higher risk of NHL compared to women who sat for fewer than three hours/day. In analyses of specific subtypes, sitting time was associated with risk of multiple myeloma only (6+ vs. 3 hours/day sitting: HR=2.40, 95% CI: 1.45-3.97). Women who engaged in any recreational physical activity had a non-significant 20-30% lower risk of NHL (p-trend=0.05) compared to women who reported no recreational physical activity. Neither leisure-time sitting nor recreational physical activity was associated with risk of NHL or major NHL subtype in men. There was no evidence of statistical interaction between physical activity and sitting time, or between body mass index and physical activity or sitting time. Further research is needed to confirm an association between sitting time and multiple myeloma and explore a possible association between physical activity and NHL.

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor (TGF)-β superfamily, has been shown to have inhibitory effect on many tumor types. However, the effect of BMP-2 on human renal cell carcinoma is still unknown. We previously showed that BMP-2 inhibits tumorigenicity of cancer stem cells in human osteosarcoma OS99-1 cells. This study investigates the effect of BMP-2 on human renal cell carcinoma using ACHN and Caki-2 cell lines. Three types of BMP receptors were found to be expressed in ACHN and Caki-2 cells. In vitro, BMP-2 was found to inhibit the growth of ACHN and Caki-2 cells. The antiproliferative effect seems to be due to cell cycle arrest in the G1 phase, which was revealed by flow cytometry analysis. Using RT-PCR analysis, we demonstrated BMP-2 up-regulated osteogenic markers Runx-2 and Collagen Type I gene expression in ACHN and Caki-2 cells. Treatment of ACHN and Caki-2 cells with BMP-2 induced a rapid phosphorylation of Smad1/5/8. In vivo, all animals receiving low number of ACHN (1×10⁴) and Caki-2 (5×10⁴) cells treated with 30 μg BMP-2 per animal showed limited tumor growth with significant bone formation, while untreated cells developed large tumor masses without bone formation in NOD/SCID mice. These results suggest that BMP-2 inhibits growth of renal cell carcinoma as well as causes induction of osseous bone formation. Further research is needed to determine the relationship between inhibition of cell proliferation and bone induction.

We performed a pooled analysis of data on self-reported history of infections in relation to the risk of non-Hodgkin lymphoma (NHL) from 17 case-control studies that included 12,585 cases and 15,416 controls aged 16-96 years at recruitment. Pooled odds ratios (OR) and 95% confidence intervals (95% CI) were estimated in two-stage random-effect or joint fixed-effect models, adjusting for age, sex and study centre. Data from the two years prior to diagnosis (or date of interview for controls) were excluded. A self-reported history of infectious mononucleosis (IM) was associated with an excess risk of NHL (OR=1.26, 95% CI=1.01-1.57 based on data from 16 studies); study-specific results indicate significant (I²=51%, p=0.01) heterogeneity. A self-reported history of measles or whooping cough was associated with an approximate 15% reduction in risk. History of other infection was not associated with NHL. We find little clear evidence of an association between NHL risk and infection although the limitations of data based on self-reported medical history (particularly of childhood illness reported by older people) are well recognised. © 2012 Wiley-Liss, Inc.

Elevated platelet counts in patients diagnosed with malignant tumors were first described more than a hundred years ago. Today it is well known that in many types of solid tumors, thrombocytosis at the time of diagnosis is associated with shorter survival. From this well documented clinical correlation between platelet count and prognosis of solid tumors the following questions arise: i.) Are the increased platelet counts the reason for shortened survival as platelet-secreted cytokines might boost tumor growth and angiogenesis? ii.) Do platelets affect tumor metastasis thereby shortening survival time? or iii.) Are increased platelet counts simply an epiphenomenon of tumor growth with larger tumors resulting in higher platelet counts and shorter survival times? We address these three questions within this review of the current literature to provide a comprehensive overview of the current concepts in tumor-platelet interaction. © 2012 Wiley-Liss, Inc.

The pathways by which Merkel cell polyomavirus (MCV) infection contributes to the formation of Merkel cell carcinomas are important for understanding the pathogenesis of these cancers. We hypothesized that MCV T antigen suppresses normal responses to ultraviolet radiation (UVR)-induced DNA damage. An MCV-infected cell line (MKL-1) exhibited UVR hypersensitivity, impaired repair of DNA lesions and cell cycle arrest following UVR, as well as reduced levels of the DNA damage recognition protein, XPC. When ectopically expressed in uninfected UISO cells, mutant but not wild-type T antigen resulted in loss of repair of UVR-induced cyclobutane pyrimidine dimers and reductions in XPC, p53 and p21 levels, whereas both wild-type and mutant T antigen inhibited cell cycle arrest following UVR. Similarly, only mutant T antigen in normal fibroblasts inhibited DNA repair and XPC expression, while both mutant and wild-type T antigens produced cell cycle dysregulation. Wild-type T antigen expression produced large T, 57kT and small T antigens while mutant T antigen was only detectable as a truncated large T antigen protein. Expression of wild-type large T antigen but not small T antigen inhibited the G1 checkpoint in UISO cells, but neither wild-type large T nor small T antigens affected DNA repair, suggesting that large T antigen generates cell cycle defects, and when mutated may also impair DNA repair. These results indicate that T antigen expression by MCV can inhibit key responses to UVR-induced DNA damage and suggest that progressive MCV-mediated abrogation of genomic stability may be involved in Merkel cell carcinogenesis. © 2012 Wiley-Liss, Inc.

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV-positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert anti-apoptotic effect in EBV-infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV-positive NPC cells. Increased apoptosis and proteolytic cleavage of PARP, caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC cells were observed. >85% of NPC cells expressing immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins co-expressed cleaved caspase-3. Tracking of expression of EBV lytic proteins and cleaved caspase-3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA effects apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer. © 2012 Wiley-Liss, Inc.

We analyzed the association between meat intake, heterocyclic amines (HCAs) and bladder cancer (BC) risk in a large case-control study comprised of 884 BC cases and 878 healthy controls, recruited from 1999 to 2009. Epidemiologic and dietary data were collected via an in-person interview. Compared to the lowest quartile of red meat intake, the odds ratios (ORs) for the second, third and fourth quartiles were 1.17 (95% CI= 0.87 to1.58), 1.47 (95% CI= 1.09 to1.99), and 1.95 (95% CI=1.41 to 2.68), respectively (P-for trend <0.001). In a subset of participants with intakes of heterocyclic amines (HCAs) available, compared to those with the lowest quartile of intake, the ORs for the second, third, and fourth quartiles were 1.47 (95% CI= 0.60 to 3.64), 2.58 (95% CI=1.09 to 6.11), and 3.32 (95% CI=1.37 to 8.01), respectively (P for trend<0.001). In cumulative analysis of SNPs in the pathway, compared to subjects carrying 0-4 unfavorable genotypes, subjects carrying 5, and 6 or more unfavorable genotypes were at 1.60-fold (95% CI= 1.20 to 2.12) and 2.37-fold (95% CI=1.82 to 3.10) increased risk, respectively. Moreover, subjects carrying six or more unfavorable genotypes and whose red meat intake was in the highest quartile were at 5.09-fold increased risk (95% CI: 2.89-8.96; P<0.001). These results strongly support that high red meat intake, high intake of HCAs and carrying high number of unfavorable genotypes in the HCA metabolic pathways are associated with increased risk of BC in the study population. © 2012 Wiley-Liss, Inc.

Nuclear factor-κB (NF-κB) is constitutively activated in many cancers, including oral squamous cell carcinoma (OSCC), and is involved in the invasive characteristics of OSCC, such as growth, anti-apoptotic activity and protease production. However, the cellular mechanism underlying NF-κB's promotion of bone invasion by OSCC is unclear. Therefore, we investigated the role of NF-κB in bone invasion by OSCC in vivo. Immnunohistochemical staining of OSCC invading bone in 10 patients indicated that the expression and nuclear translocation of p65, a main subunit of NF-κB, was increased in OSCC compared with normal squamous epithelial cells. An active form of p65 phosphorylated at serine 536 was present mainly in the nucleus in not only differentiated tumor cells but also tumor-associated stromal cells and bone-resorbing osteoclasts. We next injected mouse OSCC SCCVII cells into the masseter region of C3H/HeN mice. Mice were treated for 3 weeks with a selective NF-κB inhibitor, NBD peptide, which disrupts the association of NF-κB essential modulator (NEMO) with IκB kinases. NBD peptide treatment inhibited TNFα-induced and constitutive NF-κB activation in SCCVII cells in vitro and in vivo, respectively. Treatment with NBD peptide decreased zygoma and mandible destruction by SCCVII cells, reduced number of osteoclasts by inhibiting RANKL expression in osteoblastic cells and SCCVII cells, increased apoptosis and suppressed the proliferation of SCCVII cells. Taken together, our data clearly indicate that inhibition of NF-κB is useful for inhibiting bone invasion by OSCC. © 2012 Wiley-Liss, Inc.

Integration of the HPV (human papillomavirus) genome into the host chromatin is a characteristic step in cervical carcinogenesis. Integration ensures constitutive expression of the viral oncogenes E6 and E7 which drive carcinogenesis. However, integration also has an impact on host DNA. There is increasing evidence that integration not only occurs in fragile sites and translocation breakpoints but also in transcriptionally active regions. Indeed, a substantial number of integration sites actually disrupt host genes and may thereby affect gene expression. No doubt, even subtle changes in gene expression may influence the cell phenotype but small fold changes are difficult to quantify reliably in biopsy material. We have therefore addressed the question whether a complete loss of gene function i.e. insertional mutagenesis in combination with deletion or epigenetic modification of the second allele is also a phenomenon pertinent to cervical cancer. Of ten pre-selected squamous cell carcinomas analyzed all viral integration sites were located within the intron sequences of known genes giving rise to viral-cellular fusion transcripts of sense orientation. Moreover, for two tumours we provide evidence for complete functional loss of the gene affected by HPV integration. Of particular note is that one of the genes involved is the recently described novel tumour suppressor gene CASZ1. Although this study provides no functional proof that any of the genes affected by HPV integration are causally involved in the transformation process an exhaustive systematic look at the role of insertional mutagenesis in cervical cancer appears to be warranted. © 2012 Wiley-Liss, Inc.

The measurement of fecal tumor M2-pyruvate kinase (PKM2), over-expressed in tumor cells, has been proposed as a novel tool for detecting colorectal cancer(CRC). However, the sensitivity and specificity of this test varied among studies. The aim of this meta-analysis was to determine the diagnostic accuracy of fecal PKM2 for CRC and to evaluate its utility in the CRC screening. It was compared with guaiac fecal occult blood test (gFOBT) or immunological fecal occult blood test (iFOBT). Through comprehensive literature search, 10 studies met the inclusion criteria and were included. Summary estimates for sensitivity and specificity were calculated by using the bivariate random effect model. The hierarchical summary receiver operating characteristic (HSROC) curve was also undertaken. The overall sensitivity and specificity of fecal PKM2 for detecting CRC were 79% (95%CI 75%-83%) and 81% (95%CI 73%-87%), respectively. The summary positive predictive value and negative predictive value were 74% (95%CI 56%-87%) and 86% (95%CI 79%-91%), respectively. The pooled diagnostic odds ratio was 16 (95%CI 10-26). In head-to-head comparison, the diagnostic odds ratio of PKM2 and gFOBT for CRC were 10.167 (95%CI 5.992-17.250) and 6.557 (95%CI 3.467-12.403),respectively. The diagnostic odds ratio of PKM2 and iFOBT for CRC were 9.542 (95%CI 5.893-15.452) and 67.248 (95%CI 16.194-279.26), respectively. The fecal PKM2 test was a diagnostic tool with moderate sensitivity and specificity for detecting CRC. Its diagnostic efficiency was similar to that of gFOBT. Due to its relatively low specificity and positive predict value, fecal PKM2 was not recommended used alone as a screening tool for CRC. © 2012 Wiley-Liss, Inc.

About 50% of children with neuroblastoma (NB) show a metastatic disease and have a poor prognosis. However, disease progression is greatly variable and depends on patient's age and MYCN oncogene amplification. To investigate the role of patient's age in tumor aggressiveness, we performed array-CGH and gene-expression profiles of three groups (G) of metastatic NBs: G1, stage 4S patients and MYCN single copy (MYCN–) tumors; G2, stage 4 patients, ≤ 18 months of age, MYCN– tumors and favorable outcome; G3, stage 4 patients ≥ 19 months with unfavorable outcome. G1 was characterized by numerical aberrations prevalently; on the contrary, all G3 tumors had structural rearrangements, while G2 showed an intermediate pattern. The average of numerical alterations decreased significantly from G1 to G2 to G3 (p<0.01). Contrarily, the number of structural aberrations increased from G1 to G2 to G3 (p<2.35E-05). Noteworthy, G3/MYCN– NBs were characterized by several complex intra-chromosome rearrangements. Expression analysis of the three groups showed significant differences in genes of Rho and Ras signaling pathways, development and adhesion, cell cycle regulation and telomerase. Accumulation of structural alterations increased with patient's age and was associated with a more aggressive disease. Abnormal expression of genes involved in cell cycle and telomerase in G3, may be responsible for the genomic instability in this cohort of patients. The higher DNA instability observed in G3/MYCN– NBs than in MYCN amplified G3 may also explain why patients ≥ 19 months have a poor outcome independently by MYCN status.

Hybrid-Capture 2 HPV DNA Test® (HC2) is FDA- approved and is a proven aid in detecting HPV infections of the cervix and as an aid in diagnosing, with cytology, cervical disease. A prospective feasibility study was conducted to determine if HC2 testing has utility when screening for high-grade anal dysplasia (AIN2+). We enrolled 298 patients (45% HIV+) who had AIN2+ screening with cytology, histology and HC2 testing for two specimens: a swab into liquid-based cytology medium and either a swab or brush collection in Specimen Transport Medium (STM). High-resolution anoscopy was performed on all patients; with biopsy of AIN2+ suspicious lesions. Cytology was benign (42%), ASCUS (30%), LSIL (18%), HSIL (1%), ASC-H (1.7%) and non-diagnostic (7%) and 36% had AIN2+histology. Sensitivity and specificity for predicting AIN2+ histology for any abnormal cytology were 77% and 52%, while HC2 sensitivity and specificity were 91% and 40% (p=0.005 for sensitivity). There was no significant difference in HC2 sensitivity or specificity between brush and swab or STM and residual cells from cytology. AIN2+ was found in 20% of patients with benign cytology. Only 9 AIN2+ specimens were HC2 negative. This prospective study indicates that HC2 may be useful when screening for anal dysplasia; a larger study is recommended.

Optical imaging is a promising technique to visualize cancer tissue during surgery. In this study, we explored the use of combinations of near-infrared fluorescence agents that emit fluorescence signal at different wavelengths and each target specific tumor characteristics. Two combinations of agents (ProSense680 combined with 2DG CW800 and MMPSense680 combined with EGF CW800) were used to detect hypopharyngeal cancer in an animal model. ProSense680 and MMPSense680 detect increased activity of cathepsins and matrix metalloproteinases, respectively. These enzymes are mainly found in the invasive tumor border due to degradation of the extracellular matrix. 2DG CW800 detects tumor cells with high glucose metabolism and EGF CW800 is internalized by the epidermal growth factor receptor of tumor cells. Whole-body imaging revealed clear demarcation of tumor tissue using all 4 agents. The tumor-to-background ratio (standard deviation, p-value) was 3.69 (0.72, p<0.001) for ProSense680; 4.26 (1.33, p<0.001) for MMPSense680; 5.81 (3.59, p=0.02) for 2DG CW800; and 4.84 (1.56, p<0.001) for EGF CW800. Fluorescence signal corresponded with histopathology and immunohistochemistry, demonstrating signal of ProSense680 and MMPSense680 in the invasive tumor border, and signal of 2DG CW800 and EGF CW800 in the tumor tissue. In conclusion, we demonstrated the feasibility of dual wavelength tumor detection using different targeting strategies simultaneously in an animal model. Combined targeting at different wavelengths allowed simultaneous imaging of different tumor characteristics. Near-infrared fluorescence optical imaging has the potential to be translated into the clinic in order to improve the complete removal of tumors by real-time image-guided surgery.

5-Methylcytosine (5mC) in genomic DNA has important epigenetic functions in embryonic development and tumour biology. 5-Hydroxymethylcytosine (5hmC) is generated from 5mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) as to investigate the presence and distribution of 5hmC in human brain and brain tumours. In the normal adult brain IHC identified 61.5 % 5hmC positive cells in the cortex and 32.4 % 5hmC in white matter (WM) areas. In tumours, positive staining of cells ranged from 1.1 % in glioblastomas (WHO grade IV) to 8.9% in grade I gliomas (pilocytic astrocytomas). In the normal adult human brain LC-MS also showed highest values in cortical areas (1.17 % 5hmC/dG [deoxyguanosine]), in the cerebral white matter we measured around 0.70 % 5hmC/dG. 5hmC levels were related to tumour differentiation, ranging from lowest values of 0.078% 5hmC/dG in glioblastomas (WHO grade IV) to 0.24 % 5hmC/dG in WHO grade II diffuse astrocytomas. 5hmC measurements were unrelated to 5mC values. We find that the number of 5hmC positive cells and the amount of 5hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells (ESC) is also associated with brain tumour differentiation and anaplasia.

Copy number variations (CNVs) of the glutathione-S-transferase theta-1 (GSTT1) and glutathione-S-transferase mu-1 (GSTM1) gene loci can lead to complete lack of enzyme and have been associated with colorectal cancer (CRC) risk. Since GSTs are involved in the detoxification of xenobiotics, CNVs may modify CRC risk associated with smoking exposure and menopausal hormone therapy (MHT) use.

We investigated CRC risk associated with GSTT1 and GSTM1 CNVs and their interaction with smoking in 1796 cases and 1806 age-, sex- and residence-matched controls from a German population-based case-control study (DACHS). The interaction with MHT was assessed in the subset of 684 postmenopausal female cases and 681 controls. Trimodular genotypes (0/0, 1/0, 1/1) were determined with relative quantification based on multiplex real-time PCR. The associations with CRC risk as well as possible effect modifications were evaluated using conditional logistic regression analysis.

CNVs of GSTT1 and GSTM1 were not significantly associated with CRC risk. Compared to the 1/1 genotype, ORs for the 0/1 genotype and the 0/0 genotype were 0.89 (95% CI: 0.77-1.04) and 0.97 (95% CI: 0.80-1.18) for GSTT1, and 0.99 (95% CI: 0.78-1.27) and 1.03 (95% CI: 0.81-1.31) for GSTM1. Compared to the non-null genotype, ORs for the null-genotype were 1.04 (95% CI: 0.87-1.23) for GSTT1 and 1.03 (95% CI: 0.91-1.18) for GSTM1. No significant interaction with smoking and MHT use was observed.

The present study does not provide evidence for a strong association between CRC risk and CNVs of GSTT1 or GSTM1 or for an effect modification of smoking or MHT use. © 2012 Wiley-Liss, Inc.

Tumor progression and metastasis are promoted by the remodeling of organized tissue architecture and engagement of molecular interactions that support tumor cell passage through endothelial barriers. Prostate tumor cells that secrete and turn over excessive quantities of pericellular hyaluronan (HA) exhibit accelerated growth kinetics and spontaneous lymph node metastasis in mice. The HA Receptor for Endocytosis (HARE) is an endocytic clearance receptor for HA in the liver that is also highly expressed in sinusoidal endothelium of lymph nodes and bone marrow, which are frequent sites of prostate cancer metastasis. In this study, we tested the hypothesis that HARE can act as an endothelial receptor for metastatic tumor cells with pericellular HA. In an orthotopic mouse model of prostate cancer, we delivered a monoclonal antibody against HARE that specifically blocks HA binding and internalization. This treatment fully blocked the formation of metastatic tumors in lymph nodes. No effects on primary tumor growth were observed and the antibody did not induce toxic outcomes in any other tissue. Our results implicate HARE for the first time in potentiation of tumor metastasis and suggest a novel mechanism by which tumor cell-associated HA could promote tissue-specific dissemination. © 2012 Wiley-Liss, Inc.

Associations between clinical outcome of cancer patients and the gene expression signature in primary tumors at time of diagnosis have been reported. To test whether gene expression patterns in non-involved lung tissue might correlate with clinical stage in lung adenocarcinoma (ADCA) patients, we compared the transcriptome of non-involved lung samples from 60 ADCA smoker patients of clinical stage I versus 60 patients with stage >I. Quantitative PCR of 10 genes with the most significant differential expression confirmed the statistical association with clinical stage in 8 genes, 6 of which were downregulated in highstage patients. Five of these 6 genes were also downregulated in lung ADCA tissue as compared to non-involved tissue. Studies in vitro indicated that 4 of the genes (SLC14A1, SMAD6, TMEM100, and TXNIP) inhibited colony formation of lung cancer cell lines transfected to overexpress the genes, suggesting their potential tumor-suppressor activity. Our findings suggest that individual variations in the transcriptional profile of non-involved lung tissue may reflect the lung ADCA patient's predisposition to tumor aggressiveness.

Recurrent chromosomal alterations are found in roughly 20% of all uterine fibroids but in the majority cytogenetic changes are lacking. Recently, mutations of the gene mediator subcomplex 12 (MED12) have been detected in a majority of fibroids but no information is available whether or not they co-occur with cytogenetic subtypes as e.g. rearrangements of the genes encoding high mobility group AT-hook (HMGA) proteins. In a total of 80 cytogenetically characterized fibroids from 50 patients we were not only able to confirm the frequent occurrence of MED12 mutations but also to stratify two mutually exclusive pathways of leiomyomagenesis with either rearrangements of MED12 reflected by clonal chromosome abnormalities affecting 12q14∼15 or by mutations affecting exon 2 of MED12. On average the latter mutations were associated with a significantly smaller tumor size. However, G>A transitions of nucleotides c.130 or c.131 correlate with a significantly larger size of the fibroids compared to other MED12 mutations thus explaining the high prevalence of the former mutations among clinically detectable fibroids. Interestingly, fibroids with MED12 mutations expressed significantly higher levels of the gene encoding wingless-type MMTV integration site family, member 4 (WNT4). Based on these findings and data from the literature we hypothesize that estrogen and the mutated MED12 cooperate in activating the Wnt pathway which in turn activates β-catenin known to cause leiomyoma-like lesions in a mouse model. The occurrence of a “fibroid-type mutation” in a rare histologic subtype of endometrial polyps suggests that this mechanism is not confined to uterine leiomyomas.

Previous human intervention trial demonstrated that vitamin E (Ve) and selenium (Se) supplementation decreased esophageal cancer deaths among younger participants, but may have no effect or produce an opposite effect among older ones. In this study, we intended to mimic this human nutritional trial to determine the chemopreventive effects of Ve/Se supplementation at the early or late stage of esophageal carcinogenesis in rats. Esophageal squamous cell carcinoma (ESCC) was induced in F344 rats with N-nitrosomethylbenzylamine (NMBzA) (0.35 mg/kg BW, s.c., 3 times/wk for 5 weeks). The rats were maintained on a modified AIN-93M diet with low levels of Ve/Se or supplemented with high levels of Ve/Se at different stages. At Week 25, the number and volume of visible tumors, the numbers of dysplasia and ESCC were significantly lower in rats of supplementation during the early stage (Group C) or during the entire experimental period (Group E), but not during the late stage (Group D). Ve/Se supplementation at early stage also significantly decreased cell proliferation, nuclear fator kappaB (NFκB) activation, protein and mRNA expression of cyclooxygenase 2 and 5-lipoxygenase, biosynthesis of prostaglandin E2 and leukotriene B4 during the carcinognenesis of rat esophagus. Our results demonstrated that the chemopreventive efficacy of Ve/Se supplementation on NMBzA-induced esophageal cancer is time selective, and supplementation during the early stage is clearly effective but probably ineffective during the late stage of carcinogenesis. NFκB signaling pathway activation and aberrant AA metabolism might be the underlying mechanism.

To study the risk of childhood acute leukemia (AL) around French nuclear power plants (NPPs).

The nationwide Geocap case-control study included the 2,753 cases diagnosed in mainland France over 2002-2007 and 30,000 contemporaneous population controls. The last addresses were geocoded and located around the 19 NPPs. The study used distance to NPPs and a dose-based geographic zoning (DBGZ), based on the estimated dose to bone marrow related to NPP gaseous discharges.

An odds ratio (OR) of 1.9 [1.0-3.3], based on 14 cases, was evidenced for children living within 5 km of NPPs, compared to those living 20 km or further away, and a very similar association was observed in the concomitant incidence study (standardized incidence ratio (SIR) = 1.9 [1.0-3.2]). These results were similar for all the 5-year age groups. They persisted after stratification for several contextual characteristics of the municipalities of residence. Conversely, using the DBGZ resulted in OR and SIR close to one in all of the dose categories. There was no increase in AL incidence over 1990-2001 and over the entire 1990-2007 period. The results suggest a possible excess risk of AL in the close vicinity of French NPPs in 2002-2007. The absence of any association with the DBGZ may indicate that the association is not explained by NPP gaseous discharges. Overall, the findings call for investigation for potential risk factors related to the vicinity of NPP, and collaborative analysis of multisite studies conducted in various countries.

The accuracy of common markers for PI3K/AKT and MAPK pathway activation in preclinical and clinical cancer biomarker studies depends on phosphoepitope stability and changes of phosphorylation under ischemia. Herein, we define conditions under which phosphoepitopespecific duplex immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tumor tissues (FFPET) reflects pathway activation in situ as accurately as possible, and identify activation patterns linked to mutational status, pathway dependency and tumor microenvironment in clinical tumor samples, cell culture and xenograft tissues. Systematically assessing robustness of pAKT, pERK1/2, pMEK1/2, and pmTOR detection and related markers in xenograft tissues exposed to ischemia, we show that control of preprocessing and ischemia times allows accurate interpretation of staining results. Phosphorylation patterns were then analyzed in 33 xenograft models and in 58 breast cancer cases, including 21 paired samples of core-needle biopsies with corresponding mastectomy specimens, and 37 mastectomy samples obtained under rigorously controlled conditions minimizing ischemia time. Patterns of pAKT and pERK1/2 staining (predominant PI3K/AKT, predominant MAPK, and concomitant activation) were associated with sensitivity to pathway inhibition and partially with the mutational status in cell lines and corresponding xenograft tumors. In contrast, no clear correlation between mutational status and staining patterns was observed in clinical breast cancer samples, suggesting that interaction with the human tumor microenvironment may interfere with the use of phosphoepitope-specific IHC as potential markers for pathway dependency. In contrast to core needle biopsies, surgically resected breast cancer samples showed evidence of severe signal changes comparable to those effects observed in xenograft tumors exposed to controlled ischemia.

CYP1A1 catalyze the conversion of polycyclic aromatic hydrocarbons (PAH) into reactive metabolites which may induce DNA damage. We hypothesized that DNA methylation of the CYP1A1 enhancer could be involved in inter-individual differences in mRNA levels of CYP1A1 or affect the smoking-induced DNA damage in human lung. Using DNA bisulfite conversion and pyrosequencing, we show that DNA methylation of the CYP1A1 enhancer is affected by smoking. In adjacent histologically normal lung from lung cancer patients (n=120), low levels of DNA methylation of the CYP1A1 enhancer were related to high levels of smoking-induced hydrophobic DNA adduct (p < 0.03), and to the presence of TP53 or K-ras mutations in the corresponding lung tumors (p < 0.03). We found an inverse correlation between DNA methylation of the CYP1A1 enhancer and mRNA levels in vivo (Spearman r = -0.54; p < 0.0001). Thus, in lung tumor tissues, the CYP1A1 enhancer hypermethylation was associated with lower mRNA levels compared to adjacent histologically normal tissue (p < 0.0001). In vitro, using a panel of cultured human lung cells, we found hypermethylation of the CYP1A1 enhancer in cancer cell lines and an inverse correlation between DNA methylation and mRNA levels (Spearman r = -0.53; p = 0.003). Altogether, our results indicated that low levels of DNA methylation of the CYP1A1 enhancer in histologically normal human lung were associated with high CYP1A1 mRNA levels and with smokinginduced genetic alterations; thus it may play a role in the initiation of lung carcinogenesis.

Currently used clinico-pathological parameters are insufficient for a reliable prediction of metastatic risk and disease-free survival (DFS) of patients with clear-cell renal cell carcinoma (ccRCC). To identify prognostic genes, the expression profiles of primary ccRCC obtained from patients with different DFS — eight synchronously, nine metachronously, and seven not metastasized tumors — were determined by genome-wide expression analyses. Synchronously and metachronously metastasized primary ccRCC differed in the expression of 167 genes. Thirty-six of these genes were also differentially expressed in synchronously vs. metachronously developed pulmonary metastases analyzed in a previous study. Due to their DFS-associated deregulation that is concordant in metastases and primary ccRCC these genes are potentially functionally involved in metastatic tumor growth and are also prognostically useful.

A prognostic impact was confirmed for the genes CD31, EDNRB, and TSPAN7 at the mRNA level (n=86), and for TSPAN7 at the protein level (n=106). Patients with a higher gene expression of EDNRB or TSPAN7, or with TSPAN7-positive vessels in both cores investigated on tissue microarrays had a significantly longer DFS and tumor-specific survival (TSS). Patients with a higher CD31 gene expression showed a significantly longer TSS. EDNRB was an independent prognostic marker for the DFS. CD31, EDNRB, and TSPAN7 had an independent impact on the TSS.

In summary, comparative analysis of primary tumors and metastases is appropriate to identify independent prognostic markers in ccRCC. Gene expression of CD31 and EDNRB, and endothelial TSPAN7 protein level are potentially useful to improve outcome prediction due to their independent prognostic impact.

Carcinogenic human papillomavirus (HPV) infections are very common after sexual debut and nearly all become undetectable (“clear”) within a few years. Following clearance, the long-term risks of type-specific HPV re-appearance and subsequent risk of cervical intraepithelial neoplasia grade 2 or worse (CIN2₊) are not well defined.

In the 7-year, population-based cohort study in Guanacaste, Costa Rica, we studied how often type-specific carcinogenic HPV infections re-appeared after clearance, and how often re-appearance led to CIN2₊. We considered 1740 carcinogenic HPV infections detected by MY09/11 PCR among 2805 women (18-91 years old, median 34) who were actively followed at 6- or 12- month intervals. We identified women with 1 or more type-specific HPV infections that cleared and re-appeared, and further defined a subgroup of “definite clearance and re-appearance” (≥ 2 intervening negative results over a period of ≥ 1 year). We determined the absolute risk of CIN2₊ among the different groups. P values are two sided.

Only 7.7% (81/1052) of HPV-infected women had intervening negative results. Very few (3.7%, 39/1052) had “definite clearance and re-appearance”, of which 5.1% (2/39) subsequently persisted to a diagnosis of CIN2. There were zero CIN3₊ lesions.

Extremely few women (2/2805 of women in our cohort) had a type-specific carcinogenic HPV infection clear, re-appear and lead to CIN2₊. If confirmed, this argues against vaccination to avoid re-appearance that leads to precursor lesions and against the need of frequent HPV screening after initial negative results.

Two genome wide association studies (GWAS) have identified 5p15.33 (TERT-CLPTM1L) as one of the susceptible regions for glioma in European background. A replication research of our group highlighted the association signals in the TERT gene of this region in a Chinese Han population. To comprehensively explore the region of glioma association at 5p15.33 and to refine the potential causal variants to a smaller critical region, we conducted a fine-mapping association study among 983 cases and 1024 controls in a Chinese Han population. Using Hapmap 3 datasets as a reference, we genotyped 16 tag SNPs across this 87.9kb region encompassing TERT. Significant association with glioma risk was observed for rs2853677 [GG vs GA: adjusted OR= 1.46, P= 5.51×10-6, GG vs AA: adjusted OR= 1.72, P=7.64×10^-6, GG vs GA and AA: adjusted OR = 1.96, P=6.8×10^-6] in TERT and an uncommon CLPTM1L haplotype G–T–A of rs4635969, rs6554759 and rs414965 (haplotype frequency = 0.07 ) was associated with higher glioma risk compared with the most common G–C–G haplotype (adjusted OR=1.44, simulated P=6.00×10^-3 under additive model).

Our results indicate that sequence variants in the region flanking rs2853677 may account for the GWAS and replication signals identified in 5p15.33 for glioma susceptibility in Chinese population; besides, haplotype G–T–A in CLPTM1L also confers a risk to glioma suggesting CLPTM1L is also involved in the etiology of glioma. Additional studies especially those taking advantage of sequencing platforms are warranted to further confirm the conclusions and go deeper with our findings.

The relationship between obesity and prostate cancer risk has been studied extensively but with inconsistent findings, particularly for tumour aggressiveness. Few studies have investigated weight change and prostate cancer incidence or mortality. Using the Melbourne Collaborative Cohort Study, which recruited 17,045 men aged between 40 to 69 years at study entry, we investigated associations between reported weight and BMI at age 18 years and measured at study entry, height, weight change between age 18 years and study entry and prostate cancer incidence and mortality. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression. During follow up (mean 15 years) of 16,514 men, we ascertained 1,374 incident prostate cancers, of which 410 were classified as aggressive, and 139 deaths from prostate cancer. The incidence of all prostate cancer was not associated with body size or weight change. Weight and BMI at study entry were positively associated with aggressive prostate cancer risk (HR=1.06, 95% CI:1.00-1.13, per 5 kg; HR=1.27, 95% CI:1.08-1.49 per 5kg/m²) and prostate cancer mortality (HR=1.12, 95% CI:1.01-1.23, per 5 kg; HR=1.49, 95% CI:1.11-2.00, per 5kg/m²). Weight gain was positively associated with prostate cancer mortality (HR 1.13, 95% CI:1.02-1.26 per 5kg increment); the HR for ≥20 kg weight gain between age 18 and study entry compared with <5 kg gain over this period was 1.84, 95% CI:1.09-3.09. Higher adult weight and BMI increases the risk of aggressive prostate cancer and mortality from prostate cancer. Weight gain during adult life is associated with increased prostate cancer mortality.

Heme oxygenase-1 (HMOX1) and bilirubin UDP-glucuronosyltransferase (UGT1A1) enzymes, both involved in bilirubin homeostasis, play an important role in the oxidative stress defense. The objective of our study was to assess the effect of promoter variations of HMOX1 and UGT1A1 genes, as well as of serum bilirubin on the risk of sporadic colorectal cancer (CRC). This exploratory case-control study was based on 777 CRC patients and 986 controls from the Czech Republic. The (GT)_n and (TA)_n dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis. In addition, the A(-413)T variant in HMOX1 promoter was also analyzed using a PCR-RFLP method. Serum bilirubin levels were compared in a subset of 174 cases and 247 controls, for whom biochemical data was available. After adjustment for age, a significant association between CRC risk and UGT1A1*28 allele carrier status was detected (OR [95% CI] = 0.80 [0.60-0.97], p=0.022). No association between CRC risk and individual HMOX1 gene variants was observed, although a diplotype analysis revealed an increased risk for a specific HMOX1 genotype combination. These effects were more pronounced in males. Substantially lower serum bilirubin levels were detected in CRC patients compared to the controls (p<0.001); each 1 μmol/L decrease in serum bilirubin was associated with a 7% increase of CRC risk (p<0.001). In conclusion, UGT1A1*28 allele carrier status might be a protective factor against the development of CRC in the male population, while low serum bilirubin levels are associated with an increased risk of CRC in both genders.

Activated Kras gene coupled with activation of Akt and NF-κB trigger the development of pancreatic intraepithelial neoplasia (PanIN), the precursor lesion for pancreatic ductal adenocarcinoma (PDAC) in humans. Therefore, intervention at premalignant stage of disease is considered an ideal strategy to delay the tumor development. Pancreatic malignant tumor cell lines are widely used, however there are not relevant cell-based models representing premalignant stages of PDAC to test intervention agents. By employing a novel Kras-driven cell-based model representing premalignant and malignant stages of PDAC, we investigated the efficacy of ACTICOA-grade cocoa polyphenol (CP) as a potent chemopreventive agent under in vitro and in vivo conditions. It is noteworthy that several human intervention/clinical trials have successfully established the pharmacological benefits of cocoa-based foods. The LC-MS/MS data confirmed epicatechin as the major polyphenol of CP. Normal, non-tumorigenic and tumorigenic pancreatic ductal epithelial (PDE) cells (exhibiting varying Kras activity) were treated with CP and epicatechin. CP and epicatechin treatments induced no effect on normal PDE cells, however caused a decrease in the (i) proliferation, (ii) GTP-bound Ras protein, (iii) Akt phosphorylation and (iv) NF-κB transcriptional activity of premalignant and malignant Kras-activated PDE cells. Further, oral administration of CP (25 mg/kg) inhibited the growth of Kras-PDE cell-originated tumors in a xenograft mouse model. LC-MS/MS analysis of the blood showed epicatechin to be bioavailable to mice after CP consumption. We suggest that (a) KRas-driven cell-based model is an excellent model for testing intervention agents, and (b) CP is a promising chemopreventive agent for inhibiting PDAC development.

Human proto-oncogene c-Jun and c-Fos assemble the AP-1 complex which is a crucial transcription factor responding to environmental factors and promotes tumorgenesis. We hypothesized that genetic variants in these two genes may alter the carriers' susceptibility to lung cancer. In two independent case-control studies, we genotyped three putative functional polymorphisms (-1318T>G and -673T>C of c-Jun; -60C>T of c-Fos) in Southern Chinese and then validated the association in Eastern Chinese. We found that compared with -1318TT genotype, the -1318GT/GG variant genotypes had an increased lung cancer risk (OR = 1.46, 95% CI= 1.26-1.69); and the -673CC genotype had an increased lung cancer risk compared to -673TT/CT genotypes (OR = 1.35, 95% CI= 1.17-1.56) in the total 1559 cases versus 1679 controls. After combined these two loci, the number of the risk genotypes was associated with increased cancer risk in a dose-response manner (P_trend=2.21×10⁻¹¹), moreover, the risk genotypes interacted with smoking or drinking status on increasing cancer risk (P value of interaction were 0.009 and 0.007, respectively). We further found that those with -1318GT/GG genotypes, -673CC genotypes or both genotypes in c-Jun had higher mRNA and protein expression levels in vivo; and those variants had higher transcription activities in reporter genes in vitro, especially under the stimuli with tobacco extract or alcohol mixture as luciferase assay shown. However, for -60C>T of c-Fos, no significant association was observed for lung cancer risk. Our data suggested that genetic variants in c-Jun (-1318T>G and -673T>C) both increase the carriers' susceptibility to lung cancer via interacted with smoking or drinking on increasing the c-Jun's expression.

Endoplasmic reticulum (ER) stress-induced cancer cell apoptosis has become a novel signaling target for the development of therapeutic drugs for cancer treatment. Curcumin, a dietary phytochemical, exhibits growth-suppressive activity against cancer cells via multi-target mechanisms. However, the low stability and poor pharmacokinetics significantly limit its clinical applications. Thus, we designed and synthesized a novel mono-carbonyl analogue of curcumin, 1,5-bis(2-methoxyphenyl)penta-1,4-dien-3-one (B63). This compound exhibited a higher chemical stability in cultural medium and a better intracellular profile than curcumin. Treatment with B63 potently induced apoptosis of human non-small cell lung cancer (NSCLC) cells in a dose-responsive manner, while exhibiting no cytotoxicity in normal lung fibroblast cells. Its anti-tumor effect was associated with the ER stress-mediated apoptotic pathway and, ultimately, the activation of the caspase cascades. However, curcumin at the same concentrations did not cause ER stress in H460 cells. Further, CHOP knockdown by siRNA attenuated B63-induced cell apoptosis, indicating that the apoptotic pathway is ER stress-dependent. In vivo, the volume and weight of the tumor were reduced significantly by pre-treating the H460 tumor cells with B63 before implantation. Taken together, these insights on the novel compound B63, from both chemical and biological perspectives, may provide a novel anti-cancer candidate for the treatment of NSCLC.

Bone tumours comprise 0.2% of cancers overall but 5.7% in 15–24 year-olds. To explore the relationship with adolescence we have analysed age-incidence patterns of bone tumours in a large national dataset. Data on incident cases of bone tumours in 0–84 year-olds in England, 1979-2003, were extracted from national cancer registration data. Incidence rates per million person-years by; 5-year age-group, sex, morphology and primary site were calculated and adjusted to the world standard population. 9146 cases were identified giving an overall age-standardized rate of 7.19 per million person-years. The distribution by morphology was: osteosarcoma, 34.2%; chondrosarcoma, 27.2%; Ewing sarcoma, 19.3%; other, 19.4%. The distribution varied by age. Ewing sarcoma was most common in 0–9 year-olds, osteosarcoma in 10–29 year-olds and chondrosarcoma in 30–84 year-olds. 29.2% of all tumours occurred in 0–24 year-olds. Highest incidence of osteosarcoma and Ewing sarcoma in females was in 10–14 year-olds. In males peak incidence occurred at 15–19 years and exceeded that in females. Chondrosarcoma incidence steadily increased with age. The proportions of Ewing sarcomas occurring in respective bones were consistent with those of the adult skeleton by weight. In osteosarcoma tumours of long bones of lower limb were markedly over-represented in the adolescent peak, being six times more than at any other site. Variation in incidence patterns with age and site suggests pubertal bone growth to be a key factor in osteosarcoma while different biological pathways could be relevant for Ewing sarcoma. © 2011 Wiley-Liss, Inc.

Little is known about any consequences of swallowing tobacco-free betel-quid (TF-BQ) juice/remnants following chewing and its carcinogenic impact on the upper aerodigestive tract (UADT) to gastrointestinal tract (GIT). We investigated the neoplastic impact of TF-BQ on different anatomical locations along UADT and GIT, and differences according to their histological categories. We conducted a multicenter case-control study examining patients with 2163 pathology-proven UADT and GIT cancers, comparing them with 2250 control subjects. Generalized additive models, piecewise regression and polytomous logistic models were applied to identify possible dose-dependent structures and cancer risks. Contrary to non-significant GIT-adenocarcinoma risk (aOR=0.9), TF-BQ users experienced a 1.7 to 16.2-fold higher risk of UADT-squamous cell carcinomas than non-users, with the peak risk discovered in oral neoplasms. We separately observed a curvilinear and linear TF-BQ dose-risk relationship in oral/pharyngeal/esophageal and laryngeal cancers. Chewers of betel inflorescence were generally at a greater UADT cancer risk. A higher first-piecewise increased risk of esophageal cancer was recognized among areca-fluid swallowers than among non-swallowers (continuous aOR=1.12 vs. 1.03). TF-BQ use accounted for 66.1-78.7% and 17.8-33.2% of the cases of oral/pharyngeal and esophageal/laryngeal cancers, respectively. However, a reduction from heavy TF-BQ consumption to low-to-moderate consumption only reduced 11.3-34.6% of etiologic fraction of oral/pharyngeal cancers. Alcohol supra-additively modified the risk of TF-BQ in determining the development of oral, pharyngeal and esophageal cancers. In conclusion, the interplay of TF-BQ and alcohol/tobacco use, combined with how chewing habit is practiced, influences carcinogenic consequences on anatomically diverse sites of UADT and GIT cancers, and histologically different types. © 2011 Wiley-Liss, Inc.

The risk for colorectal cancer may be influenced by the dietary intake of various vitamins, minerals and essential fatty acids. We conducted a pooled analysis of dietary data collected using food diaries in seven prospective studies in the United Kingdom Dietary Cohort Consortium. 565 cases of colorectal cancer were matched with 1,951 controls on study centre, age, sex and recruitment date. Dietary intakes of retinol, vitamin A, thiamin, riboflavin, vitamin B6, folate, vitamin B12, vitamin D, calcium, iron, magnesium, potassium, n-6 fatty acids, n-3 fatty acids and the ratio of n-6 to n-3 fatty acids were estimated and their associations with colorectal cancer examined using conditional logistic regression models, adjusting for exact age, height, weight, energy intake, alcohol intake, fiber intake, smoking, education, social class and physical activity. There were no statistically significant associations between colorectal cancer risk and dietary intake of any of the vitamins, minerals or essential fatty acids examined. © 2011 Wiley-Liss, Inc.

Endometrial serous adenocarcinoma (ESC) is aggressive and carries a poor prognosis. p53 is frequently mutated in ESC. microRNAs (miRNAs) are a direct p53 target and have been implicated in cancer cell behavior. In the present study, we compared miRNA expression levels in ESC with the levels in endometrial endometrioid adenocarcinoma (EEC) and normal endometria. Six miRNAs were identified as having aberrant downregulation specific to ESC with miR-34b being most pronounced. miR-34b was found to have promoter hypermethylation, which when reversed, restored miR-34b expression in the cell lines treated with 5-aza-2' deoxycytidine (DAC). Ectopic expression of miR-34b in turn inhibited cell growth, migration, and most notably invasion. Our findings suggest a relationship between p53 mutation, miR-34b promoter methylation, and tumor cell behavior. These effects are likely mediated by the downstream target of miR-34b, the proto-oncogene MET, a known prognostic factor in endometrial carcinomas. The expression of MET was reduced following the restoration of miR-34b in cell lines. In summary, our data suggest that miR-34b plays a role in the molecular pathogenesis of endometrial cancer. © 2011 Wiley-Liss, Inc.

Local failure at the primary site is a major problem after chemoradiotherapy (CRT) in patients with esophageal squamous cell carcinoma (ESCC). Salvage surgery is the only treatment option with curative intent, but it is associated with high morbidity and mortality. The aim of this study was to evaluate the efficacy and safety of salvage photodynamic therapy (PDT) after CRT. Patients with histologically proven local failure limited to the submucosal layer, and without any metastasis after definitive CRT (> 50 Gy) for ESCC were enrolled in the study. PDT began with intravenous administration of 2 mg/kg of porfimer sodium followed 48-72 hours later by excimer dye laser irradiation with a fluence of 75 J/cm². The primary endpoint was a complete response (CR) to treatment with PDT, and the secondary endpoints were toxicity related to PDT, progression-free survival (PFS), and overall survival (OS). Twenty-five patients were enrolled in the study. A CR was attained in 19 of 25 patients treated with PDT (CR rate, 76%; 95% CI, 55%–91%). One treatment-related death (4%) caused by gastrointestinal hemorrhage at the irradiated site occurred 33 days after PDT. No adverse events greater than grade 3 were related to PDT in the other patients. After a median follow-up of 48 months after PDT, the PFS and OS at 3 years were 40% (95% CI, 21%–59%) and 38% (95% CI, 17%–60%), respectively. PDT is a potentially curative and tolerable salvage treatment after CRT for carefully selected patients with local failure without any metastasis.

Resistance to chemotherapeutic agents constitutes a major problem in the treatment of cancer. Over the past years, multi targeted protein kinase inhibitors such as Gleevec, Sunitinib and Sorafenib are gaining wider acceptance for cancer treatment. These drugs show anti-tumor activity in vitro and in patients. Extended usage of these drugs in therapy commonly results in disease progression due to formation of resistance caused by rearrangements and accumulation of mutations in the unstable cancer cell genome. However, the underlying drug-specific mechanisms for the development of resistance remain elusive. Hence, a detailed understanding of the molecular genetic events involved in this processes is pivotal to counteract drug-resistance. In this study, we selected for a model system of parental, Sunitinib- and Sorafenib resistant kidney carcinoma- and melanoma cell lines for comparative gene-expression analyses. We report that the degree of Sunitinib- but not Sorafenib resistance strongly correlates with increased PRKX, TTBK2 and RSK4 expression. The specific reduction of these genes employing siRNA was sufficient to sensitize the kidney- and melanoma cell lines against Sunitinib. In line with the elevated expression of PRKX, TTBK2 or RSK4, this sensitization effect was strikingly higher in the Sunitinib resistant cell lines, suggesting an expression based mechanism of these genes to trigger Sunitinib resistance. Hence, we propose that PRKX, TTBK2 and RSK4 are potential resistance markers in Sunitinib therapy and might therefore represent targets for the development of novel strategies to overcome resistance. © 2011 Wiley-Liss, Inc.

The host immune response plays a major role in colorectal carcinoma (CRC) progression. A mechanism of tumor immune escape might involve expression of the human leucocyte antigen (HLA)-E/β2m on tumor cells. The inhibitory effect of HLA-E/β2m on CD8+ cytotoxic T lymphocytes (CTL) and natural killer (NK) cells is mediated by the main HLA-E receptor CD94/NKG2A. As the pathophysiological relevance of this mechanism in CRC remains unknown, this prompted us to examine, in situ, in a series of 80 CRC 1) the HLA-E and β2m co-expression by tumor cells, 2) the density of CD8+, cytotoxic, CD244+ and NKP46+ intraepithelial tumor-infiltrating lymphocyte (IEL-TIL) and 3) the expression of CD94/NKG2 receptor on IEL-TIL. These data were then correlated to patient survival. We provided 1) the in situ demonstration of HLA-E/β2m over-expression by tumor cells in 21% of CRC characterized by an over-representation of signet ring cell carcinomas, mucinous carcinomas, and medullary carcinomas, 2) the significant association between HLA-E/β2m over-expression by tumor cells and increased density of CD8+ cytotoxic, CD244+, and CD94+ IEL-TIL, and finally 3) the unfavourable prognosis associated with HLA-E/β2m over-expression by tumor cells. Our findings show that HLA-E/β2m over-expression is a surrogate marker of poor prognosis, and point to a novel mechanism of tumor immune escape in CRC in restraining inhibitory IEL-TIL.

Untransformed HTLV-1 positive CD4⁺ cells from infected individuals are selected for expressing tax and displaying morphological features consistent with telomere dysfunctions. We show that in resting HTLV-1 positive CD4⁺ cells cloned from patients, hTERT expression parallels tax expression and cell cycling. Upon activation, these cells dramatically augment tax expression whereas their increase in telomerase activity is about twenty times lower than that of their uninfected counterpart. Activated HTLV-1 positive CD4⁺ but not uninfected CD4⁺ or CD8⁺ clones also repress the transcription of TRF1, TPP1, TANK1, POT1, DNA-PKc, and Ku80. Both infected and uninfected lymphocytes from infected individuals shared common telomere gene deregulations toward a pattern consistent with premature senescence. ATLL cells displayed the highest telomerase activity (TA) whereas recovered a telomere gene transcriptome close to that of normal CD4⁺ cells. In conclusion HTLV-1-dependent telomere modulations seem involved in clonal expansion, immunosuppression, tumor initiation and progression. © 2011 Wiley-Liss, Inc.

We observed previously that two carbohydrate epitopes, extended type 1 chain Le^a-Le^a and Le^b-Le^a, are expressed strongly in human gastric or colorectal cancer and cell lines derived therefrom, but their expression in human normal colorectal cells is highly limited. A monoclonal antibody, termed GNX-8, was established through immunization of “KM mice” with colonic cancer cell line Colo205, and with purified Le^b-Le^a glycosphingolipid, followed by screening human IgG directed to this antigen. KM mice possess human chromosome fragments and are capable of producing human immunoglobulin. GNX-8 reacted specifically with extended type 1 chain epitope Le^b-Le^a, bound to all five colonic cancer cell lines so far tested, and displayed strong complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The antigens defined by GNX-8, expressed in Colo205 cells, were: (i) glycosphingolipids with epitope Le^b-Le^a, whose reactivity was abolished upon defucosylation; (ii) glycoproteins with molecular mass range from 32 to >175 kDa, which were depleted in cells cultured in the presence of benzyl-α-GalNAc, indicating that these epitopes are O-linked glycans.

Immunohistological reactivity of GNX-8 at 1 μg/ml, applied on tissue sections from colorectal and various other types of cancer, was much stronger than that with various normal cells and tissues. GNX-8 reactivity with normal cells required a much higher concentration (150 μg/ml), and this reactivity was based on cross-reaction with non-extended, normal blood group Le^b antigen. Growth of subcutaneous xenograft of human colonic cancer cells, Colo205 or DLD-1, in nude mice or SCID mice, was strongly inhibited by administration of GNX-8. These observations, taken together, indicate that antibody GNX-8, directed specifically to Le^b-Le^a antigen, provides a novel direction of immunotherapy for human colorectal cancer. © 2009 UICC

Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray™ platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing.

The aim of our study was to evaluate the efficacy and safety of liver transplantation in patients with cholangiocarcinoma. According to the requirements of Cochrane systematic review, a thorough literature search was performed in PubMed/Medline, Embase and Cochrane electronic databases between 1995 and 2009 in terms of the key words “liver transplantation” and “cholangiocarcinoma,” “cholangiocellular carcinoma” or “bile duct cancer,” with restricted articles for the English language. Data were processed for a meta-analysis by Stata 10 software. Altogether 14 clinical trials containing 605 transplanted patients of bile duct cancers were finally enrolled in our study. The overall 1-, 3- and 5-year pooled survival rates were 0.73 [95% confidence interval (CI) = 0.65–0.80], 0.42 (95% CI = 0.33–0.51) and 0.39 (95% CI = 0.28–0.51), respectively. Of note, preoperative adjuvant therapies [orthotopic liver transplantation (OLT)-PAT group] rendered the transplanted individuals with comparably favorable outcomes with 1-, 3- and 5-year pooled survival rates of 0.83 (95% CI = 0.57–0.98), 0.57 (95% CI = 0.18–0.92) and 0.65 (95% CI = 0.40–0.87). In addition, the overall pooled incidence of complications was 0.62 (95% CI = 0.44–0.78), among which that of OLT-PAT group (0.58; 95% CI = 0.20–0.92) was relatively acceptable compared to those of liver transplantation alone (0.61; 95% CI = 0.33–0.85) and liver transplantation with extended bile duct resection (0.78; 95% CI = 0.55–0.94). In comparison to curative resection of cholangiocarcinoma with the 5-year survival rate reported from 20 to 40%, the role of liver transplantation alone is so limited. In the future, attention will be focused on liver transplantation following neoadjuvant radiochemotherapy, which requires a well-designed, prospective randomized controlled study.

ABO blood type has been associated with risk and survival for several malignancies; however, data for an association with breast cancer are inconsistent. Our study population consisted of Nurses' Health Study participants with self-reported serologic blood type and/or ABO genotype. Using Cox proportional hazards regression, we examined the association between serologic blood type and incident breast cancer among 67,697 women, including 3,107 cases. In addition, we examined the association with ABO genotype in a nested case-control study of 1,138 invasive breast cancer cases and 1,090 matched controls. Finally, we evaluated the association between serologic blood type and survival among 2,036 participants with breast cancer. No clear association was seen between serologic blood type or ABO genotype and risk of total breast cancer, invasive breast cancer or breast cancer subtypes. Compared to women with blood type O, the age-adjusted incidence rate ratios for serologic blood type and total breast cancer were 1.06 (95% CI, 0.98–1.15) for type A, 1.06 (95% CI, 0.93–1.22) for AB and 1.08 (95% CI, 0.96–1.20) for B. In genetic analyses, odds ratios for invasive breast cancer were 1.05 (95% CI, 0.87–1.27) for A/O, 1.21 (95% CI, 0.86–1.69) for A/A, 0.84 (95% CI, 0.56–1.26) for A/B, 0.84 (95% CI, 0.63–1.13) for B/O and 1.17 (95% CI, 0.35–3.86) for B/B, compared to O/O. No significant association was noted between blood type and overall or breast cancer-specific mortality. Our results suggest no association between ABO blood group and breast cancer risk or survival.

Diabetes is a risk factor for many cancers; chronic hyperglycemia is hypothesized to be, in part, explanatory. We evaluated the association between glycated hemoglobin, a time-integrated glycemia measure, and cancer incidence and mortality in nondiabetic and diabetic men and women. We conducted a prospective study of 12,792 cancer-free participants attending the second visit (1990–1992) of the Atherosclerosis Risk in Communities (ARIC) Study. We measured glycated hemoglobin in whole-blood samples using HPLC. Incident cancers were ascertained from registries and hospital records through 2006. We estimated multivariable-adjusted hazard ratios (HR) of cancer incidence and mortality for nondiabetic participants with values ≥5.7% (elevated), nondiabetic participants with <5.0% (low) and diabetic participants all compared with nondiabetic participants with 5.0–5.6% (normal). We ascertained 2,349 incident cancer cases and 887 cancer deaths. Compared with nondiabetic women with normal glycated hemoglobin, nondiabetic women with elevated values had an increased risk of cancer incidence (HR:1.24; 95% CI:1.07,1.44) and mortality (HR:1.58; 95% CI:1.23,2.05) as did diabetic women (incidence, HR:1.30; 95% CI:1.06,1.60, mortality, HR:1.96; 95% CI:1.40,2.76). Nondiabetic women with low values also had increased risk. Diabetic women with good glycemic control (<7.0%) had a lower cancer risk than those with higher values. Glycated hemoglobin in nondiabetic and diabetic men, and diabetes were not statistically significantly associated with total cancer risk. Our findings support the hypothesis that chronic hyperglycemia, even in the nondiabetic range, increases cancer risk in women. Maintaining normal glycated hemoglobin overall, and good glycemic control among diabetic adults, may reduce the burden of cancer, especially in women.