Carcinogenesis - Advance Access

Genetic Mapping of Mom5, a novel modifier of ApcMin-induced intestinal tumorigenesis

2009-07-02

The initial purpose of this study was to assess the role of ERβ in intestinal tumorigenesis by examining the effects of an estrogen receptor β knockout (ERβ^–/–) on Apc^Min mice. In order to accomplish this goal on a uniform genetic background, we were required to backcross the ERβ knockout from the 129P2 genetic background to the B6 genetic background for 10 generations. Mid way through this process, we performed a test cross in which mice from the N₅ backcross generation of the ERβ knockout strain were intercrossed with Apc^Min/+ mice to obtain Apc^Min/+ ERβ^+/+, Apc^Min/+ ERβ^+/– and Apc^Min/+ ERβ^–/– mice. Intestinal tumorigenesis in the N₅F₂ mice was evaluated at 14 weeks of age. The analysis of the impact of ERβ in the N5 cross was complicated by segregating 129P2-derived alleles that affected tumor number and were unlinked to ERβ. Genetic linkage analysis of this cross permitted the localization of a single genetic modifier of tumor number in Apc^Min/+ mice. This locus, Modifier of Min 5 (Mom5), maps to proximal mouse chromosome 5; the 129P2 allele of this locus is associated with a 50% reduction in mean intestinal tumor number. Through in silico analysis and confirmatory sequencing, we have identified the Rint-1 gene as a strong candidate for Mom5.

Activation of Thromboxane A2 Receptors Induces Orphan Nuclear Receptor Nurr1 Expression and Stimulates Cell Proliferation in Human Lung Cancer Cells

Li, X., Tai, H.-H. — 2009-07-01

Previous studies implicate that activation of thromboxane A₂ receptor (TP) induced cell proliferation and transformation in several cell lines. We report here that the activation of TP by its agonist, I-BOP, induced Nurr1 expression and stimulated proliferation of human lung cancer cells. Nurr1, an orphan nuclear receptor in the NR4A subfamily, has been implicated in cell proliferation, differentiation, and apoptosis. I-BOP markedly induced Nurr1 mRNA and protein levels as compared to other subfamily members, Nur77 and Nor-1. The signaling pathways of I-BOP-induced Nurr1 expression were examined by using various inhibitors of signaling molecules. The induction of Nurr1 expression by I-BOP appeared to be mediated through PKA/CREB, PKC and MAPK/ERK pathways and not related to EGFR and PGE₂ pathways. Transcriptional activation of Nurr1 gene by I-BOP was further investigated at the promoter level in H157 cells. 5’-Deletion analysis, site-directed mutagenesis, and luciferase reporter assay demonstrated that Nurr1 expression was induced by I-BOP in a PKA/CREB-dependent manner. Further studies have revealed that Nurr1 may mediate cyclin D1 expression and I-BOP-induced cell proliferation in H157 cells since small interfering RNA (siRNA) of Nurr1 blocked I-BOP-induced cyclin D1 expression and cell proliferation and also decreased cell growth rate. These results provide strong evidence that Nurr1 plays a significant role in cell proliferation and may mediate TP agonist-induced proliferation in lung cancer cells.

Benzyl isothiocyanate mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of MAPK in human pancreatic cancer cells

Sahu, R. P., Zhang, R., Batra, S., Shi, Y., Srivastava, S. K. — 2009-06-23

In our previous studies, we have shown that BITC inhibits the growth of human pancreatic cancer cells by inducing apoptosis. In the present study, we demonstrate the activation of all the three MAPK family members (ERK, JNK and P38) in response to BITC treatment. Exposure of Capan-2 cells with varying concentrations of BITC for 24h resulted in the phosphorylation (activation) of ERK at Thr202/Tyr204, JNK at Thr183/Tyr185 and P38 at Thr180/Tyr182, leading to the induction of apoptosis. Similar MAPK activation was also observed in Mia-Paca-2 cells in response to BITC treatment. However, normal human pancreatic ductal epithelial cells did not show the activation of MAPK's and remained unaffected by BITC treatment. To confirm the role of ERK, JNK and P38 in BITC-induced G2/M arrest and apoptosis, Capan-2 cells were pretreated with MAPK specific inhibitors or MAPK8-shRNA prior to BITC treatment. Significant protection from BITC induced G2/M arrest was observed in the cells pretreated with MEK-1 but not JNK or P38 inhibitors. On the other hand, BITC induced apoptosis was almost completely abrogated in the cells pretreated with MEK-1, JNK or P38 inhibitors. Similarly, MAPK8-shRNA also offered almost complete protection against BITC-induced G2/M arrest and apoptosis. Furthermore, we observed that BITC treatment leads to the generation of ROS in Capan-2 and MiaPaca-2 cells, which in part was orchestrated by depletion of reduced glutathione level. Blocking ROS generation with NAC significantly prevented glutathione depletion and activation of ERK and JNK but not P38. Further, NAC or tiron prevented G2/M arrest by blocking G2/M regulatory proteins and completely protected the cells from BITC-induced apoptosis. Taken together our results suggest that BITC mediated G2/M arrest is mediated through ERK activation whereas apoptosis is via ERK, JNK and P38.

Involvement of AdipoR receptor in adiponectin-induced motility and {alpha}2{beta}1 integrin up-regulation in human chondrosarcoma cells

2009-06-23

Chondrosarcoma is a type of highly malignant tumor with a capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and is involved in energy homeostasis. However, the effect of adipinectin on migration activity in human chondrosarcoma cells is mostly unknown. We found adiponectin increased the migration and expression of 2β1 integrin in human chondrosarcoma cells. The protein and mRNA expression of adiponectin receptor (AdipoR1 and AdipoR2) in chondrosarcoma patients and chondrosarcoma cell lines were significantly higher than the normal cartilage. Moreover, primary chondrosarcoma and chondrosarcoma cell lines (SW1353 and JJ012) were more invasive than normal chondrocytes. Adiponectin-mediated migration and integrin expression was attenuated by 5'-AMP-activated protein kinase (AMPK) small interference RNA and an AMPK inhibitor (araA and compound C). Activation of p38 and NF-B pathways after adiponectin treatment was demonstrated, and adiponectin-induced expression of integrins and migration activity was inhibited by the specific inhibitor and mutant of p38 and NF-B cascades. This study showed for the first time that adiponectin mediates the migration of human chondrosarcoma cells. One mechanism underlying adiponectin directed migration was transcriptional up-regulation of 2β1 integrin and activation of AdipoR receptor, AMPK, p38 and NF-B pathways

Dual role of Ski in pancreatic cancer cells: Tumor-promoting versus metastasis-suppressive function

2009-06-22

Ski used to be defined as an oncogene that contributes to the resistance of tumor cells to transforming growth factor-β (TGF-β)-induced growth arrest. As TGF-β has a dual effect on tumor growth with both tumor suppressing and promoting activity depending on the stage of carcinogenesis and the cell type, the precise role of Ski in carcinogenesis remains unclear. In this study, we show that downregulation of Ski through lentivirus-mediated RNA interference decreases tumor growth both in vitro and in vivo, yet promotes cell invasiveness in vitro, and lung metastasis in vivo in the pancreatic cancer cell line SW1990, which contain wild-type Smad4 expression, and the BxPC3 line, which is Smad4 deficient. We also show that the downregulation of Ski increases TGF-β-induced transcriptional activity, which is associated with increased TGF-β-dependent Smad2/3 phosphorylation, and results in an altered expression profile of TGF-β-inducible genes involved in metastasis, angiogenesis and cell proliferation and epithelial-mesenchymal transition. Immunohistochemical analysis of specimens from 71 patients with pancreatic adenocarcinoma showed a significant association between overexpression of Ski and decreased patient survival time (P = 0.0024). Our results suggest that Ski may act as a tumor proliferation promoting factor or as a metastatic suppressor in human pancreatic cancer.

Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor 2-mediated hepatocellular carcinoma cell invasion

2009-06-22

The expression of proteinase-activated receptor-2 (PAR₂) in human hepatocellular carcinoma (HCC) was established by reverse-transcriptase-PCR, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures, and HCC tumour tissue. Stimulation of HCC cells with trypsin and the PAR₂-selective activating peptide, 2-furoyl-LIGRLO-NH₂, increased cell invasion across Matrigel. Both effects were blocked by a PAR₂-selective pepducin antagonist peptide (pal-PAR₂) and by PAR₂ silencing with specific siRNA. PAR₂-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by down regulation of Met with specific siRNA. The involvement of Met in PAR₂-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR₂-selective agonist peptide, 2-furoyl-LIGRLO-NH₂, stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 MAPKinases was found to be critical for the PAR₂-Met receptor tyrosine kinase invasive signaling axis in HCC cells. Our study establishes an important link between the proteinase-activated receptor 2 and Met receptor tyrosine kinase signaling in promoting hepatocellular carcinoma cell invasion.

Effect of folic acid supplementation on the progression of colorectal aberrant crypt foci

Lindzon, G. M., Medline, A., Sohn, K.-J., Depeint, F., Croxford, R., Kim, Y.-I. — 2009-06-18

Whether or not folic acid supplementation promotes the progression of colorectal preneoplastic lesions to cancer is an important public health issue, given mandatory fortification and widespread supplemental use of folic acid in North America. We investigated the effect of folic acid supplementation on the progression of aberrant crypt foci (ACF), the earliest precursor of colorectal cancer. Male Sprague-Dawley rats (n = 152) were placed on a control diet (2mg folic acid/kg diet) at weaning and ACF were induced by azoxymethane. Six weeks post-ACF induction, rats were randomized to receive 0, 2, 5 or 8 mg folic acid/kg diet. At 34 weeks of age, rats were sacrificed, and colorectal tumor paremeters, plasma folate and homocysteine (a sensitve inverse indicator of tissue folate status) concentrations, and rectal epithelial proliferaiton were determined. Although the number of ACF increased as dietary folic acid levels increased (p = 0.015), the incidence of colorectal tumors did not differ significantly among the 4 dietary groups. However, tumor multiplicity was positively correlated with dietary folic acid levels (r = 0.32; p = 0.002) and inversely with plasma homosyteine concentrations (r = -0.32; p = 0.005). Tumor burden was positively correlated with dietary folic acid levels (r = 0.35; p = 0.001) and plasma folate concentrations (r = 0.33; p = 0.008) and inversely with plasma homocysteine concentrations (r = -0.42; p<0.001). Rectal epithelial proliferation was positively correalted with dietary folic acid levels (r=0.39; p<0.001) and plasma folate concentrations (r=0.34; p<0.001) and inversely with plasma homocysteine concentrations (r= -0.37; p<0.001). Our data suggest that folic acid supplementation may promote the progression of ACF to colorectal tumors.

Interferon-beta treatment increases human papillomavirus early gene transcription and viral plasmid genome replication by activating interferon regulatory factor (IRF)-1

2009-06-18

Interferons (IFNs) have been used to treat mucosal lesions caused by human papillomavirus (HPV) infection, such as intraepithelial precursor lesions to cancer of the uterine cervix, genital warts or recurrent respiratory papillomatosis (RRP), to potentially reduce or eliminate replicating HPV plasmid genomes. Mucosal HPVs have evolved mechanisms that impede IFN-β synthesis and downregulate genes induced by interferon. Here we show that these HPV types directly subvert a cellular transcriptional response to IFN-β as a potential boost in infection. Treatment with low levels of human IFN-β induced initial amplification of HPV-16 and HPV-11 plasmid genomes and increased HPV-16 or HPV-31 DNA copy numbers up to six-fold in HPV-immortalized keratinocytes. IFN treatment also increased early gene transcription from the major early gene promoters in HPV-16, HPV-31 and HPV-11. Furthermore, mutagenesis of the viral genomes and ectopic IRF expression in transfection experiments using IRF-1^-/-, IRF-2^-/- and dual knockout cell lines determined that these responses are due to the activation of interferon regulatory factor (IRF)-1 interaction with a conserved interferon response element demonstrated in several mucosal HPV early gene promoters. Our results provide a molecular explanation for the varying clinical outcomes of interferon therapy of papillomatoses and define an assay for the modulation of the HPV gene program by IFNs as well as other cytokines and signalling molecules in infection and therapy.

Increased skin carcinogenesis in caspase-activated Dnase knockout mice

2009-06-18

Caspase-activated Dnase(CAD), also called DNA fragmentation factor(DFF), is the enzyme responsible for DNA fragmentation during apoptosis, a hallmark of programmed cell death. CAD/DFF has been shown to suppress radiation-induced carcinogenesis by preventing genomic instability in cells. In this study, we have investigated the role of CAD in chemical carcinogenesis using CAD-null mice and two-stage model of skin carcinogenesis. After topical treatment of mouse skin with dimethylbenzanthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoting agent, there was a 4 fold increase in the number of papillomas per mouse and 50.8% increase in the incidence of papilloma formation in the CAD-knockout mice compared with wild type littermates. The papillomas in CAD-null mice grew faster and reached larger sizes. These data indicate that loss of CAD function enhances tumorigenesis induced by a chemical carcinogen in the DMBA/TPA two-stage model of skin carcinogenesis in mice.

Chromosome 9 Arm-Specific Telomere Length and Breast Cancer Risk

Zheng, Y.-L., Loffredo, C. A., Shields, P. G., Selim, S. — 2009-06-17

BACKGROUND: Telomere dysfunction is involved in the development of breast cancer and very short telomeres are frequent genetic alterations in breast tumors. However, the influence of telomere lengths of specific chromosomal arms on the breast cancer risk is unknown. METHODS: We conducted a case-control study of breast cancer to examine the associations of the telomere length on chromosome 9 short arms (9p) and long arms (9q) with risk of breast cancer. Chromosome 9 arm specific telomere lengths were measured by quantitative fluorescent in situ hybridization (FISH) using cultured blood lymphocytes. RESULTS: Telomere length on chromosome 9p was significantly shorter in breast cancer patients than in control subjects (P < 0.001). Using the 50^th percentile value in controls as a cut point, women who have short 9p telomeres had an increased risk of breast cancer (adjusted odds ratio [OR] = 2.6; 95% confidence interval [CI], 1.5 - 4.3). When the 9p telomere length was divided into quartiles, a significant inverse dose-response relationship between 9p telomere length and breast cancer risk was observed (P_trend < 0.001), with a quartile ORs of 3.0 (95% CI, 1.2-7.5), 3.9 (95% CI, 1.6-9.5), and 6.6 (95% CI, 2.8-15.9) for third, second and first quartile respectively when compared with women in the forth quartile. CONCLUSIONS: Short telomere length on chromosome 9p is strongly associated with the risk of breast cancer. If confirmed by future studies, chromosome 9p telomere length has the potential to be incorporated into the current prediction models to significantly enhance breast cancer risk prediction.

Differential repetitive DNA methylation in Multiple Myeloma molecular subgroups

2009-06-16

Multiple Myeloma (MM) is characterized by a wide spectrum of genetic changes. Global hypomethylation of repetitive genomic sequences such as long interspersed nuclear elements-1 (LINE-1), Alu and satellite alpha (SAT-) sequences has been associated with chromosomal instability in cancer. Methylation status of repetitive elements in MM has never been investigated. In the present study we used a quantitative bisulfite-PCR pyrosequencing method to evaluate the methylation patterns of LINE-1, Alu and SAT- in 23 human myeloma cell lines (HMCLs) and purified bone marrow plasma cells from 53 newly diagnosed MM patients representative of different molecular subtypes, 7 plasma cell leukemias (PCLs), and 11 healthy controls. MMs showed a decrease of Alu (median: 21.1%5mC), LINE-1 (70.0%5mC) and SAT- (77.9%5mC) methylation levels compared with controls (25.2%5mC, 79.5 %5mC and 89.5%5mC, respectively). Methylation levels were lower in PCLs and HMCLs compared with MMs (16.7 and 14.8%5mC for Alu; 45.5 and 42.4%5mC for LINE-1; and 33.3 and 43.3%5mC for SAT-, respectively). Notably, LINE-1 and SAT- methylation was significantly lower in the nonhyperdiploid vs hyperdiploid MMs (p=0.01 and 0.02, respectively), whereas Alu and SAT- methylation was significantly lower in MMs with t(4;14) (p=0.02 and 0.004, respectively). Finally, we correlated methylation patterns with DNA methyltranferases (DNMTs) mRNA levels showing in particular a progressive and significant increase of DNMT1 expression from controls to MMs, PCLs and HMCLs (p < 0.001). Our results indicate that global hypomethylation of repetitive elements is significantly associated with tumor progression in MM and may contribute toward a more extensive stratification of the disease.

Association of chromosome 8q variants with prostate cancer risk in Caucasian and Hispanic Men

2009-06-15

Genotyping of a 615kb region within 8q24 with 49 haplotype tagged single nucleotide polymorphisms (SNPs) in 2109 samples (797 cases and 1312 controls) of two ethnic/racial groups found SNPs which are significantly associated with the risk for prostate cancer (PCa). The highest significance in Caucasian men was found for rs6983267; the AA genotype reduced the risk for PCa (OR = 0.48, 95%CI = 0.35-0.65, p = 2.74x10^-6). This SNP also had a significant independent effect from other SNPs in the region in this group. In Hispanic men, rs7837328 and rs921146 showed independent effects (OR = 2.55, 95%CI = 1.51-4.31, p = 4.33x10^-4, OR = 2.09, 95%CI = 1.40-3.12, p = 3.13x10^-4, respectively). Significant synergist effects for increasing numbers of high-risk alleles were found in both ethnicities. Haplotype analysis revealed major haplotypes, containing the non-risk alleles, conferred protection against PCa. We found high linkage disequilibrium between significant SNPs within the region and SNPs within the Cub and Sushi Multiple Domains 1 gene (CSMD1), on the short arm of chromosome 8 in both ethnicities. These data suggest that multiple interacting SNPs within 8q24, as well as different regions on chromosome 8 far beyond this 8q24 candidate region, may confer increased risk of PCa. This is the first report to investigate the involvement of 8q24 variants in the susceptibility for PCa in Hispanic men.

{alpha}-Keto acid metabolites of organoselenium compounds inhibit histone deacetylase activity in human colon cancer cells

Nian, H., Bisson, W. H., Dashwood, W.-M., Pinto, J. T., Dashwood, R. H. — 2009-06-15

Methylselenocysteine (MSC) and selenomethionine (SM) are two organoselenium compounds receiving interest for their potential anti-cancer properties. These compounds can be converted to β-methylselenopyruvate (MSP) and -keto--methylselenobutyrate (KMSB), -keto acid metabolites that share structural features with the histone deacetylase (HDAC) inhibitor butyrate. We tested the organoselenium compounds in an in vitro assay with human HDAC1 and HDAC8; whereas SM and MSC had little or no activity up to 2 mM, MSP and KMSB caused dose-dependent inhibition of HDAC activity. Subsequent experiments identified MSP as a competitive inhibitor of HDAC8, and computational modeling supported a mechanism involving reversible interaction with the active site zinc atom. In human colon cancer cells, acetylated histone H3 levels were increased during the period 0.5-48 h after treatment with MSP and KMSB, and there was dose-dependent inhibition of HDAC activity. The proportion of cells occupying G2/M of the cell cycle was increased at 10-50 µM MSP and KMSB, and apoptosis was induced, as evidenced by morphological changes, Annexin V staining, and increased cleaved caspase-3, -6, -7, -9, and poly(ADP-ribose)polymerase. P21WAF1, a well-established target gene of clinically-used HDAC inhibitors, was increased in MSP- and KMSB-treated colon cancer cells at both the mRNA and protein level, and there was enhanced P21WAF1 promoter activity. These studies confirm that in addition to targeting redox-sensitive signaling molecules, -keto acid metabolites of organoselenium compounds alter HDAC activity and histone acetylation status in colon cancer cells, as recently observed in human prostate cancer cells.

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

2009-06-11

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

Overexpression of MUC15 Activates Extracellular Signal-Regulated Kinase 1/2 and Promotes the Oncogenic Potential of Human Colon Cancer Cells

2009-06-11

Mucins play a key role in tumorigenesis. MUC15 is a membrane-bound mucin and the MUC15 mRNA has been detected in various organs. However, its role in tumor malignancy is still unclear. This study was to investigate the MUC15 expression in colorectal tumors and the role of MUC15 in colon cancer cells. We found that the mRNA expression of MUC15 was significantly higher in 70.8% (51/72) of colorectal tumors compared with their normal counterparts by real-time RT-PCR. Immunohistochemistry showed that MUC15 expression was increased in 82.6% (43/52) of colorectal tumors. MUC15 overexpression in HCT116 cells enhanced cell proliferation, cell-extracellular matrix adhesion, colony-forming ability, and invasion. Furthermore, these effects were significantly reversed by knockdown of MUC15 with short-hairpin RNA (shRNA). In nude mice models, MUC15 overexpression significantly (P < 0.01) enhanced tumor growth. In addition, treatment of PD98059 significantly (P < 0.01) inhibited MUC15-enhanced invasion, suggesting that the invasion induced by MUC15 in HCT116 cells was primarily mediated through activation of Extracellular Signal–Regulated Kinase (ERK) 1/2. In conclusion, these results suggest that MUC15 is up-regulated in colorectal tumors and its expression enhances the oncogenic potential of colon cancer cells.

Disruption of Estrogen Receptor Signaling Enhances Intestinal Neoplasia in ApcMin/+ Mice

2009-06-11

Estrogen Receptors (ER or ESR1) and β (ERβ or ESR2) are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ER and ERβ is lost, suggesting that loss ER function might promote colorectal carcinogenesis. Through crosses between an ER knockout and Apc^Min mouse strains, we demonstrate that ER deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc^Min/+ mice. Within the normal intestinal epithelium of Apc^Min/+ mice, ER deficiency is associated with an accumulation of nuclear β-catenin, an indicator of activation of the Wnt-β-catenin signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ER deficiency is associated with activation of Wnt-β-catenin signaling, ER deficiency in the intestinal epithelium of Apc^Min/+ mice also correlated with increased expression of Wnt-β-catenin target genes. Through crosses between an ERβ knockout and Apc^Min mouse strains, we observed some evidence that ERβ deficiency is associated with an increased incidence of colon tumors in Apc^Min/+ mice. This effect of ERβ deficiency does not involve modulation of Wnt-β-catenin signaling. Our studies suggest that ER and ERβ signaling modulate colorectal carcinogenesis, and ER does so, at least in part, by regulating the activity of the Wnt-β-catenin pathway.

Epidermal Growth Factor A61G Gene Polymorphism, Gastroesophageal Reflux Disease, and Esophageal Adenocarcinoma Risk

2009-06-11

Background: Single nucleotide polymorphisms (SNPs) of key cancer genes, such as EGF A61G, are associated with an elevated risk of EAC. As GERD is an established risk factor for EAC, we evaluated whether the association between EGF polymorphism and EAC development is altered by the presence of GERD.

Methods: EGF genotyping of DNA samples was performed and GERD history was collected for 309 EAC patients and 275 matched healthy controls. Associations between genotypes and EAC risk were evaluated using adjusted logistic regression. Genotype-GERD relationships were explored using analyses stratified by GERD history and joint effects models that considered severity and duration of GERD symptoms.

Results: EGF variants (A/G or G/G) were more common (p = 0.02) and GERD was more prevalent (p<0.001) in cases than in controls. When compared to the EGF wild type A/A genotype, the G/G variant was associated with a substantial increase in EAC risk among individuals with GERD (OR 9.7; 95% CI, 3.8-25.0; p<0.001) and a slight decrease in risk for GERD-free individuals (OR 0.4; 95% CI, 0.22-0.90; p=0.02). In the joint effects models, the odds of EAC was also highest for G/G patients (when compared with A/A) who either experienced frequent GERD of greater than once per week (OR 21.8; 95% CI, 5.1-94.0; p<0.001) or suffered GERD for longer than 15 years (OR 22.4; 95% CI, 6.5-77.6; p<0.001). There was a highly significant interaction between the G/G genotype and the presence of GERD (p<0.001).

Conclusions: EGF A61G polymorphism may alter EAC susceptibility through an interaction with GERD.

Novel Single Nucleotide Polymorphism Associations with Colorectal Cancer on Chromosome 8q24 in African and European Americans

2009-06-11

Regions on chromosome 8q24 harbor susceptibility alleles for multiple cancers including colorectal (region 3) and prostate cancer (regions 1-4). The objectives of the present study were: 1) to test whether SNPs in region 4 are associated with colorectal cancer in European or African Americans, 2) to test whether 8q24 SNPs previously shown to be associated with colorectal and prostate cancer also show association in our multi-ethnic series, and 3) to test for association between 100 ancestry informative markers (AIMs) and colorectal cancer in both the African American and European American cohorts. In total, we genotyped nine markers on 8q24 and 100 unlinked AIMs in 569 colorectal cancer cases and 439 controls (490 European Americans and 518 African Americans) obtained retrospectively from a hospital-based sample. We found rs7008482 in 8q24 region 4 to be significantly associated with colorectal cancer in European Americans (p = 0.03). Also in region 4, we found that a second SNP, rs16900305, trended toward association with colorectal cancer in African Americans. rs6983267 in region 3, previously implicated in colorectal cancer risk, trended toward association with disease in European Americans but not in African Americans. Finally, none of the 100 AIMs tested for association reached statistical significance after correction for multiple hypothesis testing. In summary, these results are evidence that 8q24 region 4 contains novel colorectal cancer-associated alleles in European and African Americans.

IFN-{gamma}-dependent type 1 immunity is crucial for immunosurveillance against squamous cell carcinoma in a novel mouse carcinogenesis model

2009-06-09

Three-methylcholanthrene (MCA)-induced sarcomas have been used as conventional tools for investigating immunosurveillance against tumor development. However, MCA-induced sarcoma is not always an ideal model for the study of the human cancer system because carcinomas and not sarcomas are the dominant types of human cancers. To resolve this problem, we established a novel and simple method to induce mouse squamous cell carcinomas (SCC). As well known, the subcutaneous injection of MCA caused the formation of sarcomas at 100% incidence. However, we here first succeeded at inducing SCC at 60% of incidence within 2 months by a single intradermal injection of MCA. Using this primary SCC model, we demonstrated the critical role of IFN--dependent type 1 immunity in immunosurveillance against SCC from the following results, (i) The incidence of SCC was accelerated in IFN--deficient mice compared with that in wild-type mice; (ii) In vivo injection of CpG-ODN caused a marked reduction in the incidence of SCC in parallel with the activation of type 1-dependent antitumor immunity; (iii) The antitumor activity of CpG-ODN was significantly decreased in IFN--deficient mice. Thus, our established MCA-induced mouse SCC model could be a powerful tool for evaluating immunosurveillance mechanisms during the development of SCC and might result in a novel strategy to address immunosurveillance mechanisms of human cancer.

Snail2 cooperates with Snail1 in the repression of vitamin D receptor in colon cancer

2009-06-05

Vitamin D receptor (VDR) mediates the antitumoral action of the active vitamin D metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). VDR expression is lost during colon cancer progression causing unresponsiveness to 1,25(OH)₂D₃ and its analogs. Previously, Snail1, an inducer of epithelial-to-mesenchymal transition (EMT), was reported to inhibit VDR expression. Here, we show that Snail2/Slug, but not other EMT inducers such as Zeb1, Zeb2, E47 or Twist1, represses VDR gene promoter. Moreover, Snail2 and Snail1 show additive repressing effect on VDR promoter. Snail2 inhibits VDR RNA and protein and blocks the induction of E-cadherin and an adhesive phenotype by 1,25(OH)₂D₃. Snail2 reduces the ligand-induced VDR transcriptional activation of a consensus response element and of the CYP24 promoter. Concordantly, Snail2 inhibits the induction of CYP24 RNA and p21^CIP1, filamin A and vinculin proteins, and the repression of c-MYC by 1,25(OH)₂D₃. Additionally, Snail2 abrogates β-catenin nuclear export and the antagonism of the transcriptional activity of β-catenin/TCF complexes by 1,25(OH)₂D₃. SNAI2 expression is upregulated in 58% of colorectal tumors and correlates inversely with that of VDR. However, VDR downregulation is higher in tumors coexpressing SNAI2 and SNAI1 than in those expressing only one of these genes. Together, these data indicate that Snail2 and Snail1 cooperate for VDR repression in colon cancer.

Urinary estrogen metabolites in women at high risk for breast cancer

Im, A., Vogel, V. G., Ahrendt, G., Lloyd, S., Ragin, C., Garte, S., Taioli, E. — 2009-06-05

Objective: This study explored whether average urinary estrogen metabolites in breast cancer high risk women can be used to identify a subgroup of women at particularly high risk to develop breast cancer, to which prevention strategies should be addressed.

Methods: The population consisted of 77 high risk women, 30 breast cancer patients, and 41 controls. All subjects answered a standardized questionnaire; height and weight, and spot urine samples were also obtained. Urine hydroxyestrogen metabolites were measured in triplicate by enzyme immunoassay, and the estrogen metabolite ratios for each individual were calculated.

Results: 2:16 OHE ratio (2-hydroxyestrone/16-alpha-hydroxyestrone) in women at high risk for breast cancer was similar to that observed in the breast cancer group (1.76 ± 2.33 vs 1.29 ± 0.80), and lower than in controls (2.47 ± 1.14; p = 0.00). At the multivariate linear regression model the 2:16 OHE ratio was significantly associated with diagnosis (p = 0.000 for both the high risk and breast cancer group versus the controls) and BMI (p = 0.005), but not with age (p = 0.604), or smoking history (p = 0.478).

Conclusions: This study suggests that lower urinary 2:16 OHE ratios are predictors of breast cancer risk. Profiling estrogen metabolites may identify women who are more likely to develop breast cancer within a population of women with known risk factors, and may help to further elucidate the clinical relevance of urinary 2:16 OHE ratios as clinical markers and prognostic indicators in this population.

Triggering of Transient Receptor Potential Vanilloid Type 1 (TRPV1) by Capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ATM-dependent manner

2009-06-05

Herein, we provide evidence on the expression of TRPV1 on human urothelial cancer cells (UC), and its involvement in the apoptosis induced by the selective agonist capsaicin (CPS).

We analyzed TRPV1 mRNA and protein expression on human UC cell lines demonstrating its progressive decrease in high grade UC cells. Treatment of RT4 cells with CPS induced cell cycle arrest in G0/G1 phase and apoptosis. These events were associated with rapid coordinated transcription of pro-apoptotic genes including Fas/CD95, Bcl-2 and caspase families and ATM/CHK2/p53 DNA damage response pathway. CPS induced Fas/CD95 up-regulation, but more importantly Fas/CD95 ligand independent, TRPV1-dependent death receptor clustering and triggering of both extrinsic and intrinsic mitochondrial dependent pathways.

Moreover, we observed that CPS activates ATM kinase that is involved in Ser15, Ser20 and Ser392 p53 phosphorylation as shown by the use of the specific inhibitor KU55933. Notably, ATM activation was also found to control up-regulation of Fas/CD95 expression and its co-clustering with TRPV1 as well as RT4 cell growth and apoptosis.

Altogether, we describe a novel connection between ATM DNA damage response pathway and Fas/CD95-mediated intrinsic and extrinsic apoptotic pathways triggered by TRPV1 stimulation on UC cells.

Effect of processed and red meat on endogenous nitrosation and DNA damage

2009-06-04

Haem in red meat stimulates the endogenous production of mutagenic nitrosocompounds (NOC). Processed meat additionally contains high concentrations of preformed NOC. In two studies, of a fresh red meat (RM) versus a vegetarian diet (6 males, 6 females), and of a nitrite preserved red meat (PM) versus a vegetarian diet (5 males, 11 females), we investigated whether processing of meat might increase colorectal cancer risk by stimulating nitrosation and DNA damage. Meat diets contained 420 g (males) or 366 g (females) meat/d. Faecal homogenates from day 10 onwards were analysed for haem and NOC and associated supernatants for genotoxicity. Means are adjusted for differences in male to female ratios between studies. Faecal NOC concentrations on vegetarian diets were low (2.6 and 3.5 mmol/g) but significantly higher on meat diets, (PM 175±19 nmol/g vs RM 185±22 nmol/g, P = 0.75). The RM diet resulted in a larger proportion of nitrosyl iron (RM 78% vs PM 54%; P<0.0001) and less nitrosothiols (RM 12% vs PM 19%; P<0.01) and other NOC (RM 10% vs PM 27%; P<0.0001). There was no statistically significant difference in DNA breaks induced by faecal water following PM and RM diets (P=0.80). However, PM resulted in higher levels of oxidized pyrimidines (P<0.05). Surprisingly, vegetarian diets resulted in significantly more faecal water-induced DNA strand breaks than the meat diets (P<0.05), which needs to be clarified in further studies. Meats cured with nitrite have the same effect as fresh red meat on endogenous nitrosation, but show increased faecal water-induced oxidative DNA damage.

Insulin-like growth factor-I receptor blockade reduces the invasiveness of gastrointestinal cancers via blocking production of matrilysin

2009-06-03

Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal cancers. We have previously shown significant therapeutic activity for recombinant adenoviruses expressing dominant negative IGF-IR (IGF-IR/dn), including suppression of tumor invasion. In this study, we sought to evaluate the mechanism of inhibition of invasion and the relationship between IGF-IR and matrix metalloproteinase (MMP) activity in gastrointestinal carcinomas. We analyzed the role of IGF-IR on invasion in 3 gastrointestinal cancer cell lines, colorectal adenocarcinoma, HT29; pancreatic adenocarcinoma, BxPC3; gastric adenocarcinoma, MKN45, using a modified Boyden Chamber method and subcutaneous xenografts in nude mice. The impact of IGF-IR signaling on the expression of MMPs, and the effects of blockade of matrilysin or IGF-IR on invasiveness were assessed using recombinant adenoviruses, a tyrosine kinase inhibitor NVP-AEW541, and antisense matrilysin. Invasive subcutaneous tumors expressed several MMPs. IGF-IR/dn reduced the expression of these MMPs, but especially matrilysin (MMP-7). IGF stimulated secretion of matrilysin and IGF-IR/dn blocked IGF-mediated matrilysin induction in 3 gastrointestinal cancers. Both IGF-IR/dn and inhibition of matrilysin reduced in vitro invasion to the same degree. NVP-AEW541 also reduced cancer cell invasion both in vitro and in murine xenograft tumors via suppression of matrilysin. Thus blockade of IGF-IR is involved in the suppression of cancer cell invasion through downregulation of matrilysin. Strategies of targeting IGF-IR may have significant therapeutic utility to prevent invasion and progression of human gastrointestinal carcinomas.

Human RIF1 encodes an anti-apoptotic factor required for DNA repair

2009-05-29

Human RIF1 (Rap1 interacting protein 1) contributes to the ATM-mediated DNA damage response against the dexterous effect of DNA lesions, and plays a critical role in the S-phase checkpoint. However, the molecular mechanisms by which human RIF1 conquers DNA aberrations remain largely unknown. We here showed that inhibition of RIF1 expression by siRNA led to defective homologous recombination-mediated DNA double-strand break repair, and sensitized cancer cells to camptothecin or staurosporine treatment. RIF1 underwent caspase-dependent cleavage upon apoptosis. We further found that RIF1 was highly expressed in human breast tumors, and its expression status was positively correlated with differentiation degrees of invasive ductal carcinoma of the breast. Our results suggest that RIF1 encodes an anti-apoptotic factor required for DNA repair and is a potential target for cancer treatment.

Decoy receptor 3, upregulated by Epstein-Barr virus latent membrane protein 1, enhances nasopharyngeal carcinoma cell migration and invasion

Ho, C.-H., Chen, C.-L., Lee, W.-Y., Chen, C.-J. — 2009-05-29

Decoy receptor 3 (DcR3), a member of tumor necrosis factor receptor superfamily, has been implicated in tumorigenesis through its abilities to modulate immune responses and induce angiogenesis. Epstein-Barr virus (EBV), a ubiquitous -herpesvirus, is associated with malignancies including nasopharyngeal carcinoma (NPC). Previous studies show that DcR3 is overexpressed in EBV-positive lymphomas and Rta, an EBV transcription activator, can upregulate DcR3 in Burkitt lymphoma cell lines. However, DcR3 expression has not been demonstrated in EBV-associated NPC nor have there been any EBV latent genes linked to DcR3 upregulation. Here we showed DcR3 was overexpressed in NPC. Higher DcR3 expression score and DcR3-positive rate were found in metastatic NPC than in primary NPC tissues, suggesting DcR3 may enhance cell metastatic potential. This hypothesis is supported by our observation that NPC HONE-1 cells overexpressing DcR3 exhibited significant higher migration and invasion abilities in vitro. We found besides Rta, EBV latent protein 1 (LMP1) can upregulate DcR3 via NF-B and PI3K signaling events. Approximate 75% of LMP1-positive NPC tissues overexpressed DcR3, suggesting LMP1 may enhance DcR3 expression in vivo. Data herein suggested that increasing DcR3 expression by LMP1 not only helps EBV-associated cancer cells gain survival advantage by preventing host immune detection, but also increases the chance of cancer metastasis by enhancing cell migration and invasion. All these DcR3-mediated events facilitate normal cells to gain cancer hallmarks.

Over-expression of EphB3 Enhances Cell-Cell Contacts and Suppresses Tumor Growth in HT-29 Human Colon Cancer Cells

Chiu, S.-T., Chang, K.-J., Ting, C.-H., Shen, H.-C., Li, H., Hsieh, F.-J. — 2009-05-29

Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in Apc^Min/+ mice and EphB-mediated compartmentalization was demonstrated to be a mechanism suppressing colorectal cancer progression; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4/ephrin-A and EphB4/ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3/ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes’ stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar, and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression reorganized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical-actin-cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased Transwell migration, increased apoptosis and Ca²⁺-dependent cell-cell adhesion), decreased mesenchymal markers (fibronectin and nuclear β-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL–Rac1, a known EMT signaling pathway. Additionally, crosstalk from Wnt signaling potentiated the restoration of epithelial cell-polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3/ephrinB interaction promotes MET by re-establishing epithelial cell-cell junctions and such an MET-promoting effect contributes to EphB3-mediated tumor suppression.

Increased PEA3/E1AF and Decreased Net/Elk-3, both Ets Proteins, Characterize Human NSCLC Progression and Regulate Caveolin-1 Transcription in Calu-1 and NCI-H23 NSCLC Cell Lines

2009-05-29

Caveolin-1 protein has been called a "conditional tumor suppressor" because it can either suppress or enhance tumor progression depending on cellular context. Caveolin-1 levels are dynamic in non-small cell lung cancer (NSCLC), with increased levels in metastatic tumor cells. We have previously shown that transactivation of an ETS cis-element enhances caveolin-1 expression in a murine lung epithelial cell line. Based on high sequence homology between the murine and human caveolin-1 promoters, we proposed that ETS proteins might regulate caveolin-1 expression in human lung tumorigenesis. We confirm that caveolin-1 is not detected in well-differentiated primary lung tumors. PEA3, a pro-metastatic ETS protein in breast cancer, is expressed at low levels in well-differentiated tumors and high levels in poorly-differentiated tumors. Conversely, Net, a known ETS repressor, is expressed at high levels in the nucleus of well-differentiated primary tumor cells. In tumor cells in metastatic lymph node sites, caveolin-1 and PEA3 are highly expressed, whereas Net is now expressed in the cytoplasm. We studied transcriptional regulation of caveolin-1 in two human lung cancer cell lines, Calu-1 (high caveolin-1 expressing) and NCI-H23 (low caveolin-1 expressing). ChIP binding assays, and siRNA experiments show that PEA3 is a transcriptional activator in Calu-1 cells and that Net is a transcriptional repressor in NCI-H23 cells. These results suggest that Net may suppress caveolin-1 transcription in primary lung tumors and that PEA3 may activate caveolin-1 transcription in metastatic lymph nodes.

Cytokine Genetic Polymorphisms and Prostate Cancer Aggressiveness

2009-05-27

Prostate cancer (PCa) is one of the most common cancers in the world. Inflammation has been described as a risk factor for PCa and dependens on the production of cytokines in response to tissue damage or the presence of stimuli that induces cellular stress. Inter-individual variation in cytokine production is partially controlled by single nucleotide polymorphisms (SNPs) that have been associated with differential production of cytokines. We have recently showed that SNP-SNP interactions of cytokine genes are associated with PCa risk. However, little is known about the association of cytokine SNPs and PCa aggressiveness. In this study we evaluated the association of 15 SNPs in five cytokine genes and aggressiveness of PCa in African- and Caucasian-American individuals. Caucasian Americans with the genotypes IL10-1082GG or IL1B+3954TT had 2.31 (95% confidence interval [CI] = 1.13-4.72) and 3.11 (95%CI = 1.20-8.06) fold risk, respectively, of developing aggressive PCa, as compared with individuals without those genotypes. We did not find any associations in the African- American group. Using Multivariate Adaptive Regression Splines (MARS) modeling for exploratory SNP-SNP interactions, our results showed that more aggressive PCa in Caucasians Americans is associated with the CT genotype at IL8-47 (OR = 3.50; 95%CI = 1.13-10.88) or combined genotypes of IL1B-511CC and IL10-1082GG (OR = 3.38; 95%CI = 1.70-6.71). Unfortunately, the same analysis could not be performed in the African Americans due to limited number of individuals. With limited sample size, the results from this study suggest that SNPs in cytokine genes may be associated with PCa aggressiveness. More extensive studies are warranted to validate our findings.

The TERT-CLPTM1L lung cancer susceptibility variant associates with higher DNA adduct formation in the lung

2009-05-22

Genome-wide association studies have provided evidence that common variation at 5p15.33 (TERT-CLPTM1L), 6p21.33 and 15q25.1 (CHRNA5-CHRNA3), influences lung cancer risk and cancer types with strong environmental risk factors. To independently validate these associations we compared 5p15.33 (rs402710, rs401681), 6p21.33 (rs4324798), and 15q25.1 (rs1051730, rs16969968 and rs8034191) genotypes in 365 non-small cell lung cancer cases and 440 controls. Consistent with published data variant genotypes of 5p15 (rs402710), 6p21 and 15q25 showed dose-dependent associations with lung cancer risk. To examine if variants influence the impact of environmental risk factors on lung carcinogenesis we studied the relationship between genotype and levels of bulky aromatic/hydrophobic DNA adducts in lung tissue adjacent to tumor from 204 lung cancer cases. The risk allele of rs402710 (TERT-CLPTM1L locus) was associated with significantly higher levels of bulky aromatic/hydrophobic DNA adducts (P=0.02). These data demonstrate a potential association between the TERT-CLPTM1L variant and levels of bulky DNA adducts measured by ³²P-postlabeling and hence a basis for susceptibility to the development of lung cancer.

Prenatal N-acetylcysteine prevents cigarette smoke-induced lung cancer in neonatal mice

Balansky, R., Ganchev, G., Iltcheva, M., Steele, V. E., De Flora, S. — 2009-05-20

Certain adult diseases may have their origin early in life, and perinatal exposures may contribute to cancers both during childhood and later in life. We recently demonstrated that mainstream cigarette smoke (MCS) induces a potent carcinogenic response in mice when exposure starts soon after birth. We also showed that the antioxidant N-acetylcysteine (NAC) prevents the extensive nucleotide and gene expression alterations that occur "physiologically" at birth in mouse lung. The present study was designed to evaluate whether administration of NAC during pregnancy may affect the yield of tumors in mice exposed to MCS, starting after birth and continuing for 120 days. The results obtained showed that, 210 days after birth, one adenoma only was detectable in sham-exposed mice. In contrast, as much as the 61.1% (33/54) of MCS-exposed mice born from untreated dams had lung tumors, including both benign tumors and bronchoalveolar carcinomas. Treatment with NAC during pregnancy strikingly inhibited the formation of benign lung tumors and totally prevented occurrence of carcinomas. In addition, prenatal NAC inhibited the MCS-induced hyperplasia of the urinary bladder epithelium. These findings demonstrate for the first time that treatment during pregnancy with an antioxidant chemopreventive agent can affect the induction of tumors consequent to exposure to a carcinogen after birth.

Phospholipase C{epsilon} promotes intestinal tumorigenesis of ApcMin/+ mice through augmentation of inflammation and angiogenesis

Li, M., Edamatsu, H., Kitazawa, R., Kitazawa, S., Kataoka, T. — 2009-05-20

Apc^Min/+ mice, carrying an inactivated allele of the APC gene, are widely used as an animal model for human colorectal tumorigenesis, where tumor environment, such as inflammation, is known to play a critical role in tumor progression. We previously demonstrated that phospholipase C (PLC), an effector of Ras and Rap small GTPases, plays a crucial role in two-stage skin chemical carcinogenesis using 12-O-tetradecanoyl-phorbor-13-acetate (TPA) as a promoter through augmentation of TPA-induced inflammation. Here, we show that Apc^Min/+ mice lacking PLC (PLC^–/–) exhibit marked resistance to spontaneous intestinal tumorigenesis compared to those with the PLC^+/+ background. Time course of the development of tumors, which are histopathologically classified into low- and high-grade adenomas with increasing dysplasia and size, and adenocarcinomas, indicates that not only the low-grade adenoma formation but also the progression to high-grade adenoma are suppressed in PLC^–/–;Apc^Min/+ mice. Low-grade adenomas of PLC^–/–;Apc^Min/+ mice exhibit accelerated apoptosis and reduced cellular proliferation. They also show marked attenuation of tumor angiogenesis and reduction in expression of vascular endothelial growth factor. In contrast, high-grade adenomas of PLC^–/–;Apc^Min/+ mice exhibit marked attenuation of tumor-associated inflammation without significant differences in apoptosis and proliferation. These results suggest that PLC plays crucial roles in intestinal tumorigenesis through two distinct mechanisms, augmentation of angiogenesis and inflammation, depending on the tumor stage.

Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase

2009-05-14

Resistance to chemotherapy is a major obstacle for successful treatment of breast cancer patients. Given that prolactin acts as an anti-apoptotic/survival factor in the breast, we postulated that it antagonizes cytotoxicity by chemotherapeutic drugs. Treatment of breast cancer cells with prolactin caused variable resistance to taxol, vinblastine, doxorubicin and cisplatin. Prolactin prevented cisplatin-induced G2/M cell cycle arrest and apoptosis. In the presence of prolactin, significantly less cisplatin was bound to DNA, as determined by mass spectroscopy, and little DNA damage was seen by -H2AX staining. Prolactin dramatically increased the activity of glutathione-S-transferase, which sequesters cisplatin in the cytoplasm; this increase was abrogated by Jak and MAPK inhibitors. Prolactin upregulated the expression of the glutathione-S-transferase µ, but not the , isozyme. A glutathione-S-transferase inhibitor abrogated antagonism of cisplatin cytotoxicity by prolactin. In conclusion, prolactin confers resistance against cisplatin by activating a detoxification enzyme, thereby reducing drug entry into the nucleus. These data provide a rational explanation for the ineffectiveness of cisplatin in breast cancer, which is characterized by high expression of both prolactin and its receptor. Suppression of prolactin production or blockade of its actions should benefit patients undergoing chemotherapy by allowing for lower drug doses and expanded drug options.

Proteomic approach to ETV5 during endometrial carcinoma invasion reveals a link to oxidative stress

2009-05-14

Endometrial cancer, the most common gynecological malignancy in western countries, is characterized by a favourable prognosis. Nonetheless, deep myometrial invasion correlates with more undifferentiated tumors, lymph-vascular invasion, node involvement and decreased global survival. We have previously described the Ets family member ERM/ETV5 specifically up-regulated in endometrial endometrioid carcinoma (EEC) associated with myometrial infiltration.

To understand the role of this transcription factor during myometrial infiltration, we analysed by two-dimension differential gel electrophoresis (2D-DIGE) technology those proteins which expression was altered in endometrial cell lines stably over-expressing ERM/ETV5. Pathway analysis pointed to actin regulation and TGF-beta and progesterone signalling as processes regulated by ERM/ETV5. In addition, we characterized the specific up-regulation of the nuclear dehydrogenase/reductase Hep27 as well as its ERM/ETV5-dependent mitochondrial localization. Further functional studies demonstrated a protective role of Hep 27 against apoptosis induced by oxidative stress.

Overall, the ETV5-related proteomic approach performed in the Hec-1A cell line reinforces a role of this transcription factor in the regulation of the migratory and invasive tumour behaviour, and points to a modulated response to oxidative stress associated with the promotion of invasion in endometrial cancer. Unraveling the molecular events in EEC associated with the initiation of tumor invasion, would represent an obvious improvement in the pursuit of rational targets for the onset of metastasis. This knowledge would also be a valuable tool for the molecular stratification of patients, since myometrial affectation determines an increase in the rate of recurrence after a first surgical treatment and a decrease in five year survival.

Altered expression of the human base excision repair gene NTH1 in gastric cancer

2009-05-04

A base excision repair enzyme, NTH1, has activity that is capable of removing oxidized pyrimidines, such as thymine glycol, from DNA. To clarify whether the NTH1 gene is involved in gastric carcinogenesis, we first examined the NTH1 expression level in eight gastric cancer cell lines, and the results showed that NTH1 expression was downregulated in all of them, including cell line AGS. Next, a comparison of excisional repair activity against thymine glycol by empty-vector-transfected AGS clones and FLAG-NTH1-expressing AGS clones showed that a low NTH1 expression level led to low capacity to repair the damaged base in the gastric epithelial cells. Reduced mRNA expression of NTH1 was also detected in 36% (18/50) of primary gastric cancers. Moreover, immunohistochemical analysis revealed that NTH1 was predominantly localized in the cytoplasm in 24% (12/50) of the primary gastric cancers in contrast to the nuclear localization in non-cancerous tissue, suggesting impaired excisional repair ability for nuclear DNA. No associations between clinicopathological factors and NTH1 expression level or localization pattern were detected in the gastric cancers. Next, we found two novel genetic polymorphisms, i.e., c.-163C>G and c.-241_-221del, in the NTH1 promoter region, and a luciferase assay showed that both were associated with reduced promoter activity. However, there were no associations between the polymorphisms and risk of gastric cancer in a gastric cancer case-control study. These findings suggested that downregulation of NTH1 expression and abnormal localization of NTH1 may be involved in the pathogenesis of a subset of gastric cancers.

Candidate gene approach evaluates association between innate immunity genes and breast cancer risk in Korean women

2009-04-16

Objectives: This study was conducted to investigate the role of common variation in innate immunity related genes as susceptibility factors to breast cancer risk in Korean women. Methods: Total 1,536 single nucleotide polymorphisms (SNPs) in 203 genes were analyzed by Illumina GoldenGate assay in 209 cases and the same numbers of controls. Both SNP and gene-based tests were used to evaluate the association with breast cancer risk. The robustness of results was further evaluated with permutation method, false discovery rate (FDR), and haplotype analyses. Results: Both SNP and gene-based analyses showed promising associations with breast cancer risk for 17 genes: OR10J3, FCER1A, NCF4, CNTNAP1, CTNNB1, KLKB1, ITGB2, ALOX12B, KLK2, IRAK3, KLK4, STAT6, NCF2, CCL1, C1QR1, MBP, and NOS1. The most significant association with breast cancer risk was observed for the OR10J3 SNP (rs2494251, p-value = 1.2x10^-4) and FCER1A SNP (rs7548864, p-value = 7.7x10^-4). Gene-based permutation and FDR p-values for OR10J3 SNP (rs2494251) with breast cancer risk were also significant (p=4x10^-5 and 0.008 respectively). Haplotype analyses supported these findings that OR10J3 and FCER1A were most significantly associated with risk for breast cancer (p=2x10^-4 and 0.004 respectively). Conclusion: This study suggests that common genetic variants in the FCER1A and OR10J3 be strongly associated with breast cancer risk among Korean women.

Enhanced genomic instabilities caused by deregulated microtubule dynamics and chromosome segregation: a perspective from genetic studies in mice

Rao, C. V., Yamada, H. Y., Yao, Y., Dai, W. — 2009-04-16

Aneuploidy is defined as numerical abnormalities of chromosomes and is frequently (>90%) present in solid tumors. In general, tumor cells become increasingly aneuploid with tumor progression. It has been proposed that enhanced genomic instability at least contributes significantly to, if not requires, tumor progression. Two major modes for genomic instability are Microsatellite Instability (MIN) and Chromosome Instability (CIN). MIN is associated with DNA-level defects (e.g. mismatch repair defects), and CIN is associated with mitotic errors such as chromosome mis-segragation. The mitotic Spindle Assembly Checkpoint (SAC) ensures that cells with defective mitotic spindles or defective interaction between the spindles and kinetochores do not initiate chromosomal segregation during mitosis. Thus, the SAC functions to protect the cell from chromosome mis-segregation and anueploidy during cell division. A loss of the SAC function results in gross aneuploidy, a condition from which cells with an advantage for proliferation will be selected. During the past several years, a flurry of genetic studies in mice and humans strongly support the notion that an impaired SAC causes enhanced genomic instabilities and tumor development. This review article summarizes the roles of key spindle checkpoint proteins (i.e. Mad1/Mad1L1, Mad2/Mad2L1, BubR1/Bub1B, Bub3/Bub3 (conventional protein name (yeast or human)/mouse protein name)) and the modulators (i.e. Chfr/Chfr, Rae1/Rae1, Nup98/Nup98, Cenp-e/Cenpe, Apc/Apc) in genomic stability and suppression of tumor development, with a focus on information from genetically engineered mouse model systems. Further elucidation of molecular mechanisms of the SAC signaling has the potential for identifying new targets for rational anti-cancer drug design.

Epigenetic therapy using the histone deacetylase inhibitor for increasing therapeutic gain in oral cancer: Prevention of radiation-induced oral mucositis and inhibition of chemical-induced oral carcinogenesis

Chung, Y.-L., Lee, M.-Y., Pui, N. N.M. — 2009-04-07

In addition to genetic changes, epigenetic aberrations also play important roles in radiation- and chemical-induced disorders and carcinogenesis. The present study investigated whether epigenetic therapy with a histone deacetylase (HDAC) inhibitor has dual benefits for radiation-induced oral mucositis and chemical-induced oral carcinogenesis, which should be treated at the same time. The HDAC inhibitor phenylbutyrate was first tested to determine if it influences DNA damage repair and survival in irradiated normal cells in vitro by investigating the patterns and dynamics of phospho-H2AX foci, Rad51 foci and phospho-H2AX/Rad51 co-localization, and using the Comet and clonogenic assays. Oral mucositis or carcinogenesis was induced in hamsters using radiation or dimethylbenzylamine (DMBA) irritation to the cheek pouch. The ability of phenylbutyrate formed in proper carriers to prevent radiation-induced oral mucositis and inhibit chemical-induced oral carcinogenesis was assessed. The treated or untreated irradiated or DMBA-irritated oral tissues or mucosal epithelia were subjected to the studies of histology, immunohistochemistry, gene expression, Comet assay, HDAC activity, or oxidative stress. We found that phenylbutyrate promoted DNA repair and survival in normal cells after radiation. Compared with blank or vehicle-treated hamsters, the irradiated mucosa treated with phenylbutyrate had significantly lower oxidative stress and TNF- expression and less severe oral mucositis of a shorter duration. A reduction of the oral tumor incidence, burden, and progression by phenylbutyrate correlated with the suppression of oncomiRs and Rad51 over-expression, the up-regulation of differentiation markers, and the decrease of intracellular HDAC activity and oxidative stress during DMBA-induced oral carcinogenesis. Thus, epigenetic therapy using the HDAC inhibitor as an adjuvant to radiotherapy for chemical-induced oral cancer may provide a promising strategy combining the prevention of radiation-induced oral mucositis and the inhibition of oral carcinogenesis.

The type III transforming growth factor-{beta} receptor negatively regulates nuclear factor-{kappa}B signaling through its interaction with {beta}-arrestin2

You, H. J., How, T., Blobe, G. C. — 2009-03-26

Transforming growth factor-β (TGF-β) increases or decreases NFB signaling in a context dependent manner through mechanisms that remain to be defined. The type III TGF-β receptor (TβRIII) is a TGF-β superfamily co-receptor with emerging roles in both mediating and regulating TGF-β superfamily signaling. We have previously reported a novel interaction of TβRIII with the scaffolding protein, β-arrestin2, which results in TβRIII internalization and downregulation of TGF-β signaling. β-arrestin2 also scaffolds interacting receptors with the MAPK and NFB signaling pathways. Here we demonstrate that TβRIII, through its interaction with β-arrestin2, negatively regulates NFB signaling in MCF10A breast epithelial and MDA-MB-231 breast cancer cells. Increasing TβRIII expression reduced NFB-mediated transcriptional activation and IB degradation, while a TβRIII mutant unable to interact with β-arrestin2, TβRIII-T841A, had no effect. In a reciprocal manner, shRNA-mediated silencing of either TβRIII expression or β-arrestin2 expression increased NFB-mediated transcriptional activation and IB degradation. Functionally, TβRIII-mediated repression of NFB signaling is important for TβRIII-mediated inhibition of breast cancer cell migration. These studies define a mechanism through which TβRIII regulates NFB signaling and expand the roles of this TβRIII superfamily co-receptor in regulating epithelial cell homeostasis.

Metabolic transformation in cancer

Tennant, D. A., Duran, R. V., Boulahbel, H., Gottlieb, E. — 2009-03-25

In 2000, Douglas Hanahan and Robert Weinberg published a review detailing the six hallmarks of cancer. These are six phenotypes that a tumour requires in order to become a fully-fledged malignancy: persistent growth signals, evasion of apoptosis, insensitivity to anti-growth signals, unlimited replicative potential, angiogenesis, and invasion and metastasis. However, it is becoming increasingly clear that these phenotypes do not portray the whole story, and that other hallmarks are necessary: one of which is a shift in cellular metabolism. The tumour environment creates a unique collection of stresses to which cells must adapt in order to survive. This environment is formed by the uncontrolled proliferation of cells, which ignore the cues that would create normal tissue architecture. As a result, the cells forming the tumour are exposed to low oxygen and nutrient levels, as well as high levels of toxic cellular waste products, which is thought to propel cells towards a more transformed phenotype, resistant to cell death and pro-metastatic.