Cancer Prevention Research recent issues

Lippman, S. M. — 2009-01-05

Retinoid Chemoprevention Trials: Cyclin D1 in the Crosshairs

Freemantle, S. J., Guo, Y., Dmitrovsky, E. — 2009-01-05

Rapamycin for Chemoprevention of Upper Aerodigestive Tract Cancers

Dennis, P. A. — 2009-01-05

Oral-Specific Chemical Carcinogenesis in Mice: An Exciting Model for Cancer Prevention and Therapy

Wong, K.-K. — 2009-01-05

Cyclin D1 and Cancer Development in Laryngeal Premalignancy Patients

2009-01-05

In a previous trial, we found that combined 13-cis-retinoic acid, IFN-, and -tocopherol more effectively reversed advanced premalignant lesions of the larynx than of the oral cavity and that cyclin D1 (CD1) G/A870 single nucleotide polymorphism correlated with cancer risk. We conducted the present trial primarily to confirm the clinical activity of the combination in advanced laryngeal premalignancy and to confirm and extend our findings on CD1, both genotype and protein expression, in association with cancer risk in this setting. Twenty-seven moderate-to-severe laryngeal dysplasia patients underwent induction with combined 13-cis-retinoic acid daily, -IFN twice weekly, and -tocopherol daily for 1 year; 14 nonprogressing patients then were randomized to maintenance fenretinide or placebo for 2 years. During induction, two patients had pathologic complete responses, six had partial responses (30% overall response rate), and five developed laryngeal cancer. There were no significant differences between maintenance fenretinide and placebo in response or cancer rates. Ten patients developed cancer overall. Twenty-four patients were evaluated for the CD1 G/A870 genotype, and 23 for pretreatment and posttreatment CD1 protein expression. Consistent with our earlier report, shorter cancer-free survival was associated with the CD1 AA/AG genotype (P = 0.05). Extending our earlier work, high CD1 expression was associated with worse cancer-free survival overall (P = 0.04) and within each CD1 genotype group. These findings support CD1 genotype and protein expression as important risk markers for laryngeal cancer and suggest future trials targeting upstream regulators of CD1 transcription.

High-Dose Fenretinide in Oral Leukoplakia

2009-01-05

We previously showed that low-dose fenretinide (200 mg/d) had limited activity in retinoid-resistant oral leukoplakia (34% response rate) possibly because serum drug levels were insufficient to induce retinoid receptor–independent apoptosis. Therefore, we designed the single-arm phase II trial reported here to investigate whether higher-dose fenretinide would improve leukoplakia response over that of our previous study. Leukoplakia patients received fenretinide (900 mg/m² twice daily) in four 3-week cycles (1 week on drug followed by 2 weeks off). At week 12, clinical responses were determined and blood samples were collected for serum drug level assessments. A planned interim futility analysis led to early trial closure after the initial 15 (of 25 planned) patients because only 3 (20%) had a partial response (stopping rule: ≤4 responses in first 16 patients). Fenretinide was well tolerated—only one grade 3 adverse event (diarrhea) occurred. Serum fenretinide levels changed from 0 (baseline) to 0.122 ± 0.093 µmol/L (week 12). In correlative in vitro studies, high-dose fenretinide inhibited the growth of head and neck cancer cells more and oral leukoplakia cells less than did lower doses of fenretinide. This result is consistent with our clinical finding that high-dose fenretinide did not improve on the historical response rate of lower-dose fenretinide in our previous oral leukoplakia trial.

Targeting Mammalian Target of Rapamycin by Rapamycin Prevents Tumor Progression in an Oral-Specific Chemical Carcinogenesis Model

Czerninski, R., Amornphimoltham, P., Patel, V., Molinolo, A. A., Gutkind, J. S. — 2009-01-05

The increased molecular understanding of cancerous growth may now afford the opportunity to develop novel therapies targeting specific dysregulated molecular mechanisms contributing to the progression of each cancer type. In this regard, the aberrant activation of Akt/mammalian target of rapamycin (mTOR) pathway is a frequent event in head and neck squamous cell carcinomas (HNSCC), thus representing a potential molecular target for the treatment of HNSCC patients. The ability to translate this emerging body of information into effective therapeutic strategies, however, has been hampered by the limited availability of animal models for oral malignancies. Here, we show that the administration in the drinking water to mice of 4-nitroquinoline-1 oxide, a DNA adduct-forming agent that serves as a surrogate of tobacco exposure, leads to the progressive appearance of preneoplastic and tumoral lesions in the tongue and oral mucosa, with 100% incidence after only 16 weeks of carcinogen exposure. Remarkably, many of these lesions evolve spontaneously into highly malignant SCCs few weeks after 4-nitroquinoline-1 oxide withdrawal. In this model, we have observed that the activation of the Akt-mTOR biochemical route represents an early event, which is already detectable in dysplastic lesions. Furthermore, we show that the inhibition of mTOR by the chronic administration of rapamycin halts the malignant conversion of precancerous lesions and promotes the regression of advanced carcinogen-induced SCCs. Together, these findings support the contribution of the mTOR signaling pathway to HNSCC progression and provide a strong rationale for the early evaluation of mTOR inhibitors as a molecular-targeted strategy for HNSCC chemoprevention and treatment.

A Pilot Study of Sampling Subcutaneous Adipose Tissue to Examine Biomarkers of Cancer Risk

2009-01-05

Examination of adipose tissue biology may provide important insight into mechanistic links for the observed association between higher body fat and risk of several types of cancer, in particular colorectal and breast cancer. We tested two different methods of obtaining adipose tissue from healthy individuals. Ten overweight or obese (body mass index, 25-40 kg/m²), postmenopausal women were recruited. Two subcutaneous abdominal adipose tissue samples were obtained per individual (i.e., right and left lower abdominal regions) using two distinct methods (method A: 14-gauge needle with incision, versus method B: 16-gauge needle without incision). Gene expression was examined at the mRNA level for leptin, adiponectin, aromatase, interleukin 6 (IL-6), and tumor necrosis factor- (TNF-) in flash-frozen tissue, and at the protein level for leptin, adiponectin, IL-6, and TNF- following short-term culture. Participants preferred biopsy method A and few participants reported any of the usual minor side effects. Gene expression was detectable for leptin, adiponectin, and aromatase, but was below detectable limits for IL-6 and TNF-. For detectable genes, relative gene expression in adipose tissue obtained by methods A and B was similar for adiponectin (r = 0.64, P = 0.06) and leptin (r = 0.80, P = 0.01), but not for aromatase (r = 0.37,P = 0.34). Protein levels in tissue culture supernatant exhibited good intra-assay agreement [coefficient of variation (CV), 1-10%], with less agreement for intraindividual agreement (CV, 17-29%) and reproducibility, following one freeze-thaw cycle (CV, >14%). Subcutaneous adipose tissue biopsies from healthy, overweight individuals provide adequate amounts for RNA extraction, gene expression, and other assays of relevance to cancer prevention research.

Chemoprevention of Colorectal Neoplasia by Estrogen: Potential Role of Vitamin D Activity

2009-01-05

Postmenopausal hormone replacement therapy lowers colon cancer incidence. In humans, the mechanism is unknown, but animal models suggest that it may involve activation of the vitamin D receptor (VDR) pathway.

The aims of our study were to determine whether estrogen intervention affects global gene expression in rectal mucosal biopsies and whether vitamin D–related genes are affected.

Estradiol was given to raise serum estradiol to premenopausal levels in 10 postmenopausal women under close nutritional control. Primary end points were expression of VDR, CYP24A1, CYP27B1, and E-cadherin in rectal mucosa by reverse transcription-PCR and examining response to estradiol by genome-wide arrays. Responses in gene expression in rectal biopsies to estrogen were determined in each subject individually and compared with a human estrogen response gene array database and a custom array in vitro–generated database.

Cluster analysis showed that subjects maintained their overall gene expression profile and that interindividual differences were greater than intraindividual differences after intervention. Eight of 10 subjects showed significant enrichment in estrogen-responsive genes. Gene array group analysis showed activation of the VDR pathway and down-regulation of inflammatory and immune signaling pathways. Reverse transcription-PCR analysis showed significant up-regulation of VDR and E-cadherin, a downstream target of vitamin D action.

These data suggest that the chemopreventive action of hormone replacement therapy on colon neoplasia results, at least in part, from changes in vitamin D activity. Evaluation of gene arrays is useful in chemopreventive intervention studies in small groups of subjects.

Estrogen Receptor-{beta} as a Potential Target for Colon Cancer Prevention: Chemoprevention of Azoxymethane-Induced Colon Carcinogenesis by Raloxifene in F344 Rats

Janakiram, N. B., Steele, V. E., Rao, C. V. — 2009-01-05

Raloxifene, selective estrogen receptor (ER) modulator, is not fully explored in colorectal cancer. In the present study we, (a) investigated the effect of raloxifene on ER-positive colon cancer HCT-116 cell growth, (b) assessed the relevance of ER-β in colon tumorigenesis, and (c) assessed the chemopreventive efficacy of raloxifene against azoxymethane (AOM)-induced colon carcinogenesis using aberrant crypt foci (ACF) as surrogate end point marker. HCT-116 cells treated with raloxifene showed a significant decrease in cell growth associated with a decrease in ER-β expression levels. AOM-induced colon adenocarcinoma showed significant up-regulation of ER-β expression at both the protein and mRNA levels compared with normal mucosa, suggesting that ER-β is positively associated with colon cancer. An assay using five different dietary dose levels (0.31, 0.62, 1.25, 2.5, or 5 ppm) of raloxifene for 6 weeks in male F344 rats found the maximum tolerated dose to be 5 ppm. To evaluate inhibitory properties of raloxifene on colonic ACF, 7-week-old rats were fed experimental diets containing 0, 0.625, 1.25, and 2.5 ppm of raloxifene. After 1 week, rats received s.c. injections of AOM, 15 mg/kg body weight, once weekly for 2 weeks. Rats continued to receive respective experimental diets and sacrificed 8 weeks after the last AOM treatment. Raloxifene given in the diet significantly inhibited AOM-induced total colonic ACF (31-40%; P < 0.001-0.0005) and multicrypt (four or more) aberrant foci (23-50%; P < 0.05-0.005) in F344 rats. Our findings suggest that ER-β acts as a colon tumor promoter and raloxifene as an antagonist to ER-β, providing protection against colon carcinogenesis.

Inflammation-Associated Serum and Colon Markers as Indicators of Dietary Attenuation of Colon Carcinogenesis in ob/ob Mice

2009-01-05

Although inflammatory cytokines and obesity-associated serum proteins have been reported as biomarkers of colorectal adenoma risk in humans, little is known of biomarkers of response to interventions that attenuate tumorigenesis. Dietary navy beans and their fractions attenuate colon carcinogenesis in carcinogen-induced genetically obese mice. We hypothesized that this attenuation would be associated with changes in inflammatory cytokines and obesity-related serum proteins that may serve as measures of efficacy. ob/ob mice (n = 160) were injected with the carcinogen azoxymethane (AOM) to induce colon cancer and randomly placed on one of four diets (control, whole navy bean, bean residue fraction, or bean extract fraction) for 26 to 28 wk. Serum was analyzed for 14 inflammation- or obesity-related proteins, and colon RNA was analyzed for expression of 84 inflammation-associated genes. Six of 14 serum proteins were increased [i.e., interleukin (IL)-4, IL-5, IL-6, IL-10, IFN, granulocyte macrophage colony-stimulating factor] in hyperplastic/dysplastic stages of colon carcinogenesis. Bean-fed mice had significantly higher monocyte chemoattractant protein-1 and lower IL-6 levels in serum. In colon mucosa, 55 of 84 inflammation-associated genes differed between AOM-induced and noninduced mice. Of the 55 AOM-induced genes, 5 were counteracted by bean diets, including IL-6 whose increase in expression levels was attenuated by bean diets in AOM-induced mice. In summary, IL-6 emerged as a serum protein that was increased in hyperplastic/dysplastic stages of colon carcinogenesis, but attenuated with bean-based diet in serum and colon mucosa. Changes in a subset of inflammation-associated serum proteins and colon gene expression may serve as response indicators of dietary attenuation of colon carcinogenesis.

Selenium and Risk of Bladder Cancer: A Population-Based Case-Control Study

2009-01-05

Emerging evidence indicates a potential role of selenium in the prevention of several types of cancer, including bladder cancer. We investigated the association between toenail selenium concentrations and bladder cancer risk in a population-based case-control study in New Hampshire. We analyzed data from 857 incidence cases diagnosed between July 1, 1994 and June 30, 2001 and 1,191 general population controls. Newly diagnosed cases of bladder cancer were identified from the New Hampshire State Cancer Registry, which operates a rapid reporting system. Controls were selected from population lists (driver's license and Medicare enrollment). We used logistic regression analyses to generate odds ratios (OR) and 95% confidence intervals (95% CI), controlling for age, sex, and pack-years of smoking and conducted separate analyses according to the intensity of p53 immunohistochemical staining of the tumor. Overall, toenail selenium concentrations were not significantly related to bladder cancer [OR Q4 versus Q1, 0.90 (95% CI, 0.68-1.19); P_trend = 0.15]. However, within specific subgroups there were inverse associations, i.e., among moderate smokers [OR, 0.61 (95% CI, 0.39-0.96); P_trend = 0.004], women [OR, 0.66 (95% CI, 0.40-1.10); P_trend = 0.11], and those with p53-positive cancers [OR Q4 versus Q1, 0.57 (95% CI, 0.34-0.94); P_trend = 0.01]. Our results indicate that selenium is not inversely related to risk of bladder cancer overall; however, they raise the possibility that selenium may be preventive in certain molecular phenotypes of tumors (e.g., p53 positive) or within certain subsets of a population (e.g., women or moderate smokers).

Growth Inhibition and Regression of Lung Tumors by Silibinin: Modulation of Angiogenesis by Macrophage-Associated Cytokines and Nuclear Factor-{kappa}B and Signal Transducers and Activators of Transcription 3

2009-01-05

The latency period for lung tumor progression offers a window of opportunity for therapeutic intervention. Herein, we studied the effect of oral silibinin (742 mg/kg body weight, 5 d/wk for 10 weeks) on the growth and progression of established lung adenocarcinomas in A/J mice. Silibinin strongly decreased both tumor number and tumor size, an antitumor effect that correlates with reduced antiangiogenic activity. Silibinin reduced microvessel size (50%, P < 0.01) with no change in the number of tumor microvessels and reduced (by 30%, P < 0.05) the formation of nestin-positive microvessels in tumors. Analysis of several proteins involved in new blood vessel formation showed that silibinin decreased the tumor expression of interleukin-13 (47%) and tumor necrosis factor- (47%), and increased tissue inhibitor of metalloproteinase-1 (2-fold) and tissue inhibitor of metalloproteinase-2 (7-fold) expression, without significant changes in vascular endothelial growth factor levels. Hypoxia- inducible factor-1 expression and nuclear localization were also decreased by silibinin treatment. Cytokines secreted by tumor cells and tumor-associated macrophages regulate angiogenesis by activating nuclear factor-B (NF-B) and signal transducers and activators of transcription (STAT). Silibinin decreased the phosphorylation of p65NF-B (ser276, 38%; P < 0.01) and STAT-3 (ser727, 16%; P < 0.01) in tumor cells and decreased the lung macrophage population. Angiopoietin-2 (Ang-2) and Ang-receptor tyrosine kinase (Tie-2) expression were increased by silibinin. Therapeutic efficacy of silibinin in lung tumor growth inhibition and regression by antiangiogenic mechanisms seem to be mediated by decreased tumor-associated macrophages and cytokines, inhibition of hypoxia-inducible factor-1, NF-B, and STAT-3 activation, and up-regulation of the angiogenic inhibitors, Ang-2 and Tie-2.

Anthocyanins in Black Raspberries Prevent Esophageal Tumors in Rats

2009-01-05

Diets containing freeze-dried black raspberries (BRB) suppress the development of N-nitrosomethylbenzylamine (NMBA)–induced tumors in the rat esophagus. Using bioassay-directed fractionation, the anthocyanins in BRB were found to be the most active constituents for down-regulation of carcinogen-induced nuclear factor-B and activator protein-1 expression in mouse epidermal cells in vitro. The present study was undertaken, therefore, to determine if the anthocyanins contribute to the chemopreventive activity of BRB in vivo. F344 rats consumed diets containing either (a) 5% whole BRB powder, (b) an anthocyanin-rich fraction, (c) an organic solvent-soluble extract (a–c each contained ~3.8 µmol anthocyanins/g diet), (d) an organic-insoluble (residue) fraction (containing 0.02 µmol anthocyanins/g diet), (e) a hexane extract, and (f) a sugar fraction (e and f had only trace quantities of anthocyanins), all derived from BRB. Animals were fed diets 2 weeks before treatment with NMBA and throughout the bioassay. Control rats were treated with NMBA only. Animals were killed at week 30, and esophageal tumors were enumerated. The anthocyanin treatments (diet groups a–c) were about equally effective in reducing NMBA tumorigenesis in the esophagus, indicating that the anthocyanins in BRB have chemopreventive potential. The organic-insoluble (residue) fraction (d) was also effective, suggesting that components other than berry anthocyanins may be chemopreventive. The hexane and sugar diets were inactive. Diet groups a, b, and d all inhibited cell proliferation, inflammation, and angiogenesis and induced apoptosis in both preneoplastic and papillomatous esophageal tissues, suggesting similar mechanisms of action by the different berry components.

The Role of Helicobacter pylori in the Spectrum of Barrett's Carcinogenesis

Fassan, M., Rugge, M., Parente, P., Tieppo, C., Rugge, M., Battaglia, G. — 2009-01-05

Correction: Adiponectin Inhibits Cancer Cell Proliferation

2009-01-05

Improving the Vision of Colonoscopy: Does the Fine Print Really Matter?

Hawk, E., Guillem, J. G. — 2008-12-08

Assessing Toxicity in Cancer Chemoprevention Trials: The Other Side of the Coin

Goodman, G. E. — 2008-12-08

Pancreatic Cancer: Translating Lessons from Mouse Models and Hereditary Syndromes

Wescott, M. P., Rustgi, A. K. — 2008-12-08

Pancreatic ductal adenocarcinoma is the overwhelmingly predominant form of pancreatic cancer and the second most common type of gastrointestinal cancer (behind colorectal cancer) in the United States. Recent exciting advances in two areas of pancreatic ductal adenocarcinoma (i.e., the development and characterization of genetically engineered mouse models and the dissection of the genetic basis of hereditary forms in families) have been illuminating. These preclinical models and clinical syndromes provide the first tangible basis for progress in screening and prevention in high-risk populations and in the development of molecular diagnostics and experimental therapeutics.

Chromoendoscopy Detects More Adenomas than Colonoscopy Using Intensive Inspection without Dye Spraying

2008-12-08

Conventional colonoscopy misses some neoplastic lesions. We compared the sensitivity of chromoendoscopy and colonoscopy with intensive inspection for detecting adenomatous polyps missed by conventional colonoscopy.

Fifty subjects with a history of colorectal cancer or adenomas underwent tandem colonoscopies at one of five centers of the Great Lakes New England Clinical Epidemiology and Validation Center of the Early Detection Research Network. The first exam was a conventional colonoscopy with removal of all visualized polyps. The second exam was randomly assigned as either pan-colonic indigocarmine chromoendoscopy or standard colonoscopy with intensive inspection lasting >20 minutes. Size, histology, and numbers of polyps detected on each exam were recorded.

Twenty-seven subjects were randomized to a second exam with chromoendoscopy and 23 underwent intensive inspection. Forty adenomas were identified on the first standard colonoscopies. The second colonoscopies detected 24 additional adenomas: 19 were found using chromoendoscopy and 5 were found using intensive inspection. Chromoendoscopy found additional adenomas in more subjects than did intensive inspection (44% versus 17%) and identified significantly more missed adenomas per subject (0.7 versus 0.2, P < 0.01). Adenomas detected with chromoendoscopy were significantly smaller (mean size 2.66 ± 0.97 mm) and were more often right-sided. Chromoendoscopy was associated with more normal tissue biopsies and longer procedure times than intensive inspection. After controlling for procedure time, chromoendoscopy detected more adenomas and hyperplastic polyps compared with colonoscopy using intensive inspection alone.

Chromoendoscopy detected more polyps missed by standard colonoscopy than did intensive inspection. The clinical significance of these small missed lesions warrants further study.

Longitudinal Assessment of Air Conduction Audiograms in a Phase III Clinical Trial of Difluoromethylornithine and Sulindac for Prevention of Sporadic Colorectal Adenomas

2008-12-08

A phase III clinical trial assessed the recurrence of adenomatous polyps after treatment for 36 months with difluoromethylornithine (DFMO) plus sulindac or matched placebos. Temporary hearing loss is a known toxicity of treatment with DFMO, thus a comprehensive approach was developed to analyze serial air conduction audiograms. The generalized estimating equation method estimated the mean difference between treatment arms with regard to change in air conduction pure tone thresholds while accounting for within-subject correlation due to repeated measurements at frequencies. Based on 290 subjects, there was an average difference of 0.50 dB between subjects treated with DFMO plus sulindac compared with those treated with placebo (95% confidence interval, –0.64 to 1.63 dB; P = 0.39), adjusted for baseline values, age, and frequencies. In the normal speech range of 500 to 3,000 Hz, an estimated difference of 0.99 dB (–0.17 to 2.14 dB; P = 0.09) was detected. Dose intensity did not add information to models. There were 14 of 151 (9.3%) in the DFMO plus sulindac group and 4 of 139 (2.9%) in the placebo group who experienced at least 15 dB hearing reduction from baseline in 2 or more consecutive frequencies across the entire range tested (P = 0.02). Follow-up air conduction done at least 6 months after end of treatment showed an adjusted mean difference in hearing thresholds of 1.08 dB (–0.81 to 2.96 dB; P = 0.26) between treatment arms. There was no significant difference in the proportion of subjects in the DFMO plus sulindac group who experienced clinically significant hearing loss compared with the placebo group. The estimated attributable risk of ototoxicity from exposure to the drug is 8.4% (95% confidence interval, –2.0% to 18.8%; P = 0.12). There is a <2 dB difference in mean threshold for patients treated with DFMO plus sulindac compared with those treated with placebo.

Delphinidin Attenuates Neoplastic Transformation in JB6 Cl41 Mouse Epidermal Cells by Blocking Raf/Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling

2008-12-08

Recent studies suggest that anthocyanidins play a pivotal role in the chemopreventive effects of fruits and vegetables. However, the underlying molecular mechanisms and cellular targets remain unknown. Neoplastic transformation of cells and inflammation are considered to be major events contributing to carcinogenesis. Here, we report that delphinidin, a major dietary anthocyanidin, inhibits tumor promoter–induced transformation and cyclooxygenase-2 (COX-2) expression in JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells by directly targeting Raf and mitogen-activated protein kinase kinase (MEK). Delphinidin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced neoplastic transformation and COX-2 expression at both the protein and transcriptional levels. The activation of activator protein-1 and nuclear factor-B induced by TPA was dose dependently inhibited by delphinidin treatment. Delphinidin strongly suppressed Raf1 and MEK1 kinase activities and subsequently attenuated TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase (ERK), p90RSK, and MSK. Although delphinidin suppressed ERK and c-Jun NH₂-terminal kinase activities, it was more effective at inhibiting Raf1 or MEK1 activities. Pull-down and competition assays revealed that delphinidin binds with Raf1 or MEK1 noncompetitively with ATP. Delphinidin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor and H-Ras, both of which are involved in the activation of Raf/MEK/ERK signaling. Together, these findings suggested that the targeted inhibition of Raf1 and MEK activities and COX-2 expression by delphinidin contribute to the chemopreventive potential of fruits and vegetables.

Gene Signaling Pathways Mediating the Opposite Effects of Prepubertal Low-Fat and High-Fat n-3 Polyunsaturated Fatty Acid Diets on Mammary Cancer Risk

2008-12-08

In rats, prepubertal exposure to low-fat diet containing n-3 polyunsaturated fatty acids (PUFA) reduces mammary cell proliferation, increases apoptosis, and lowers risk of mammary tumors in adulthood, whereas prepubertal high-fat n-3 PUFA exposure has opposite effects. To identify signaling pathways mediating these effects, we performed gene microarray analyses and determined protein levels of genes related to mammary epithelial cell proliferation. Nursing female rats and rat pups were fed low-fat (16% energy from fat) or high-fat (39% energy from fat) n-3 or n-6 PUFA diets between postnatal days 5 and 24. cDNA gene expression microarrays were used to identify global changes in the mammary glands of 50-day-old rats. Differences in gene expression were confirmed by real-time quantitative PCR, and immunohistochemistry was used to assess changes in the peroxisome proliferator–activated receptor and cyclin D1 levels. DNA damage was determined by 8-hydroxy-2'-deoxyguanosine assay. Expressions of the antioxidant genes thioredoxin, heme oxygenase, NADP-dependent isocitrate dehydrogenase, and metallothionein III, as well as peroxisome proliferator–activated receptor protein, were increased in the mammary glands of 50-day-old rats prepubertally fed the low-fat n-3 PUFA diet. Prepubertal exposure to the high-fat n-3 PUFA diet increased DNA damage and cyclin D1 protein and reduced expression of BRCA1 and cardiotrophin-1. Reduction in mammary tumorigenesis among rats prepubertally fed a low-fat n-3 PUFA diet was associated with an up-regulation of antioxidant genes, whereas the increase in mammary tumorigenesis in the high-fat n-3 PUFA fed rats was linked to up-regulation of genes that induce cell proliferation and down-regulation of genes that repair DNA damage and induce apoptosis.

Tamoxifen Prevents Premalignant Changes of Breast, but not Ovarian, Cancer in Rats at High Risk for Both Diseases

Ting, A. Y., Kimler, B. F., Fabian, C. J., Petroff, B. K. — 2008-12-08

Women at increased risk for breast cancer are at increased risk for ovarian cancer as well, reflecting common risk factors and intertwined etiology of the two diseases. We previously developed a rat model of elevated breast and ovarian cancer risk, allowing evaluation of dual-target cancer prevention strategies. Tamoxifen, a Food and Drug Administration–approved breast cancer chemoprevention drug, has been shown to promote ovarian cysts in premenopausal women; however, the effect of tamoxifen on ovarian cancer risk is still controversial. In the current experiment, Fischer 344 rats (n = 8 per treatment group) received tamoxifen (TAM) or vehicle (control) in factorial combination with combined breast and ovarian carcinogen (17β-estradiol and 7,12 dimethylbenza[a]anthracene, respectively). Mammary and ovarian morphologies were normal in the control and TAM groups. Carcinogen (CARC) treatment induced mammary dysplasia with elevated cell proliferation and reduced estrogen receptor- expression and promoted preneoplastic changes in the ovary. In the CARC + TAM group, tamoxifen reduced preneoplastic changes and proliferation rate in the mammary gland, but not in the ovary, compared with rats treated with carcinogen alone. Putative stem cell markers (Oct-4 and aldehyde dehydrogenase 1) were also elevated in the mammary tissue by carcinogen and this expansion of the stem cell population was not reversed by tamoxifen. Our study suggests that tamoxifen prevents early progression to mammary cancer but has no effect on ovarian cancer progression in this rat model.

Grape Seed Extract Inhibits Angiogenesis via Suppression of the Vascular Endothelial Growth Factor Receptor Signaling Pathway

Wen, W., Lu, J., Zhang, K., Chen, S. — 2008-12-08

Blockade of angiogenesis is an important approach for cancer treatment and prevention. Vascular endothelial growth factor (VEGF) is one of the most critical factors that induce angiogenesis and has thus become an attractive target for antiangiogenesis treatment. However, most current anti-VEGF agents often cause some side effects when given chronically. Identification of naturally occurring VEGF inhibitors derived from diet would be one alternative approach with an advantage of known safety. Grape seed extract (GSE), a widely used dietary supplement, is known to have antitumor activity. In this study, we have explored the activity of GSE on VEGF receptor and angiogenesis. We found that GSE could directly inhibit the kinase activity of purified VEGF receptor 2, a novel activity of GSE that has not been characterized. GSE could also inhibit the VEGF receptor/mitogen-activated protein kinase–mediated signaling pathway in endothelial cells. As a result, GSE could inhibit VEGF-induced endothelial cell proliferation and migration as well as sprout formation from aorta ring. In vivo assay further showed that GSE could inhibit tumor growth and tumor angiogenesis of MDA-MB-231 breast cancer cells in mice. Consistent with the in vitro data, GSE treatment of tumor-bearing mice led to concomitant reduction of blood vessel density and phosphorylation of mitogen-activated protein kinase. Depletion of polyphenol with polyvinylpyrrolidone abolished the antiangiogenic activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the antiangiogenic activity. Taken together, this study indicates that GSE is a well-tolerated and inexpensive natural VEGF inhibitor and could potentially be useful in cancer prevention or treatment.

Identification of Mucin Depleted Foci in the Human Colon

2008-12-08

Aberrant crypt foci (ACF) originally described in rodents treated with colon-specific carcinogens have been identified also in humans at high risk of colon cancer (CRC) and are extensively used as cancer biomarkers. However, studies documenting the heterogeneity of ACF have questioned their precancerous nature. Recently, we described dysplastic foci depleted of mucins (MDF) in the colon of rats treated with colon-specific carcinogens. Like colon tumors, MDFs show activation of Wnt signaling driven by mutations in the β-catenin gene and Apc, a key gene in colorectal carcinogenesis. Because MDFs have been identified thus far only in rodents, we wanted to search for similar lesions in humans. Familial adenomatous polyposis (FAP) subjects, carrying germ-line mutations in the APC gene, are at high risk of CRC. Therefore, we first searched for MDF-like lesions in unsectioned colon samples from FAP patients and then in patients with sporadic CRC. MDFs were present in the colon of FAP patients (average of 0.0577 lesions/cm²) and at a much lower density in CRC patients (average of 0.0006 lesions/cm²). ACFs were also observed in all patients. Histologic preparations of all the MDFs identified in FAP and CRC consisted of microadenomas at variable grades of dysplasia. The occurrence of MDF-like lesions in high-risk patients provides evidence that these lesions have a counterpart in human pathology and, as observed in rodents, may represent the very early stages of CRC.

Dose-Dependent Inhibition of Tobacco Smoke Carcinogen-Induced Lung Tumorigenesis in A/J Mice by Indole-3-Carbinol

Kassie, F., Matise, I., Negia, M., Upadhyaya, P., Hecht, S. S. — 2008-12-08

Recently, we reported inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus benzo(a)pyrene (BaP)–induced lung tumorigenesis in A/J mice by indole-3-carbinol (I3C; 112 µmol/g diet) administered beginning at 50% in the carcinogen treatment phase. In this study, we examined the dose-dependent and postcarcinogen tumor-inhibitory activities of I3C. A mixture of NNK plus BaP (2 µmol each) administered by gavage as eight biweekly doses caused 21.1 ± 5.2 lung tumors per mouse. Carcinogen-treated mice given diets containing I3C at 1, 10, 30, 71, and 112 µmol/g, beginning at 50% in the carcinogen treatment phase, had 17.9 ± 6.1, 10.4 ± 3.7, 9.8 ± 5.1, 5.2 ± 4.0, and 2.5 ± 2.4 lung tumors per mouse, corresponding to reductions by 15%, 51%, 53%, 75%, and 88%, respectively. All reductions, except at the lowest dose level (1 µmol I3C/g diet), were significant (P < 0.001). Similarly, administration of I3C (112 µmol/g diet) beginning 1 week after the last dose of the carcinogen significantly reduced NNK plus BaP-induced lung tumor multiplicity to 5.6 ± 3.5, corresponding to a reduction by 74%. Analyses of cell proliferation and apoptosis markers revealed that I3C reduced the number of Ki-67–positive cells and expression of proliferating cell nuclear antigen, phospho-Akt, and phospho-BAD and increased cleavage of poly(ADP-ribose) polymerase, suggesting that the lung tumor inhibitory effects of I3C were mediated, at least partly, through inhibition of cell proliferation and induction of apoptosis. These results clearly show the efficacy of I3C in the prevention of tobacco carcinogen–induced lung tumorigenesis in A/J mice and provide a basis for future evaluation of this compound in clinical trials as a chemopreventive agent for current and former smokers.

A Novel Derivative of the Natural Agent Deguelin for Cancer Chemoprevention and Therapy

2008-12-08

The natural compound deguelin has promising preventive and therapeutic activity against diverse cancers by directly binding to heat shock protein-90 and thus suppressing its function. Potential side effects of deguelin over a certain dose, however, could be a substantial obstacle to its clinical use. To develop a derivative(s) of deguelin with reduced potential side effects, we synthesized five deguelin analogues (SH-02, SH-03, SH-09, SH-14, and SH-15) and compared them with the parent compound and each other for structural and biochemical features; solubility; and antiproliferative effects on normal, premalignant, and malignant human bronchial epithelial (HBE) and non–small-cell lung cancer (NSCLC) cell lines. Four derivatives destabilized hypoxia-inducible factor-1 as potently as did deguelin. Reverse-phase protein array (RPPA) analysis in H460 NSCLC cells revealed that deguelin and the derivatives suppressed expression of a number of proteins including heat shock protein-90 clients and proteins involved in the phosphoinositide 3-kinase/Akt pathway. One derivative, SH-14, showed several features of potential superiority for clinical use: the highest apoptotic activity; no detectable influence on Src/signal transducer and activator of transcription signaling, which can promote cancer progression and is closely related to pathogenesis of Parkinson's disease (deguelin, SH-02 and SH-03 strongly activated this signaling); better aqueous solubility; and less cytotoxicity to immortalized HBE cells (versus deguelin) at a dose (1 µmol/L) that induced apoptotic activity in most premalignant and malignant HBE and NSCLC cell lines. These collective results suggest that the novel derivative SH-14 has strong potential for cancer chemoprevention and therapy, with equivalent efficacy and lesser toxicity (versus deguelin).

Acknowledgement to Reviewers

2008-12-08

Abstract A1: The zinc-finger E-box-binding transcriptional repressor Snail promotes tumor progression and angiogenesis in non-small cell lung cancer.

2008-12-22

The zinc-finger E-box-binding transcriptional repressor Snail has been implicated in tumor progression of several malignancies. Best known as a transcriptional repressor of the adherens junction component E-cadherin, Snail has predominantly been associated with the epithelial-mesenchymal transition (EMT), invasion, and metastasis. However, the role of Snail in non-small cell lung cancer (NSCLC) is not yet defined. Immunohistochemistry of human lung adenocarcinoma and squamous cell carcinoma sections revealed specific nuclear staining of tumor cells in all patient samples. A human lung adenocarcinoma cell line H441 was stably transduced with a Snail expressing retroviral vector. Western analysis verified up-regulation of Snail and down-regulation of E-cadherin in the Snail over-expressing cells (H441-Snail) as compared to vector control cells (H441-V). In three-dimensional spheroid culture, H441V cells grew into large, tight spheroids while H441-Snail cells were markedly discohesive, reflecting a Snail-mediated phenotypic change consistent with EMT. To examine the effects of Snail over-expression in vivo, severe combined immunodeficiency (SCID) mice were injected subcutaneously with either H441-Snail cells or with H441-V cells. Mice were sacrificed 6 weeks later. The primary tumor burden in mice bearing H441-Snail tumors was five fold greater than the primary tumor burden of mice bearing H441-V tumors (p<0.005). To evaluate the incidence of metastases, these cell lines were tested in an orthotopic model. H441-V and H441-Snail cells were transthoracically injected into the left lung of SCID mice. Organs were then harvested and analyzed by flow cytometry gated on the human marker, CD49b. The incidence of metastases to the right lung, liver, bone marrow, and adrenal glands were significantly increased in the mice bearing H441-Snail tumors (p<0.05). Because H441-Snail tumors appeared grossly more hemorrhagic as compared to H441-V tumors at the time of harvest, primary tumors were homogenized and analyzed by ELISA for levels of two angiogenic factors known to play a role in NSCLC tumor angiogenesis: CXCL8 and CXCL5. H441-Snail tumors were associated with increased levels of CXCL8 (p<0.05) and CXCL5 (p<0.05) as compared to H441-V tumors. Based on these results, SCID mice injected with H441-Snail cells were treated with a CXCR2 (CXCL8 and CXCL5 receptor) blocking antibody. These mice exhibited reduced tumor burden and metastases as compared to mice bearing H441-Snail tumors that were treated with a control antibody. In summary, Snail contributes to tumor progression in NSCLC by inducing tumor angiogenesis, as evidenced by elevated levels of angiogenic factors (CXCL8 and CXCL5) and the reversal of increased tumor burden and metastases with CXCR2 blockade.

Citation Information: Cancer Prev Res 2008;1(7 Suppl):A1.

Abstract B1: Genome-wide LOH and copy number alterations show heterogeneity in esophageal squamous cell carcinoma

2008-12-22

Background

Genomic instability appears to play an important role in most human cancers. To more fully characterize genomic instability and assess its effect on gene expression in esophageal squamous cell carcinoma (ESCC), we examined loss of heterozygosity (LOH), copy number (CN) loss, and CN gain, as well as gene expression, using genome-wide methods in cases from a high-risk region of China.

Methods

Micro-dissected tumor and matched germ-line DNA from 30 ESCC patients from Shanxi Province, PR China who underwent surgical resection were tested using the Affymetrix GeneChip Human Mapping 500K array set and frequencies of LOH and CN alterations were determined. Matched micro-dissected tumor/non-tumor samples were also tested for RNA expression with Affymetric Human Genome U133A 2.0 arrays on a subset of 17 of these patients.

Results

Genomic instability measures varied widely among cases and separated them into two distinct groups: a high frequency instability group (two-thirds of all cases with 1 or more instability measure >10%) and a low frequency instability group (one-third of cases with no instability measures >10%). Genomic instability also varied widely across chromosomal arms, with the highest frequency of LOH on 9p (33% of informative SNPs), of CN loss on 3p (33%), and of CN gain on 3q (48%). Genome-wide LOH measured with the 500K chip produced remarkably similar frequency rankings for chromosome arms as previous less-dense genome-wide scans that used microsatellites or the 10K SNP chip, although absolute frequencies were reduced. Twelve regions with particularly high CN gain were identified, including six on 3q, one on 7q, four on 8q, and one on 11q. The most gene-rich of these CN gain regions was 11q13.1-13.4, where we identified 26 genes with frequent CN gain which also had RNA expression data available. CN gain was significantly correlated with increased RNA expression in over 80% of these genes.

Conclusions

Genomic instability patterns show two heterogeneous groups of ESCC cases; the high-frequency instability group may harbor germ-line variants or acquired somatic mutations in genes that maintain genomic stability. Genome-wide studies from this high-risk population show a consistent pattern of high LOH on selected chromosome arms which are targets in searching for loss-of-function genes involved in ESCC. Our findings demonstrate the potential utility of combining CN and gene expression data to screen for genes involved in esophageal carcinogenesis. Future studies should combine results in tumors with germ-line genotypes to find functional changes, and determine if these changes are associated with genetic susceptibility to ESCC or might serve as early detection markers.

Citation Information: Cancer Prev Res 2008;1(7 Suppl):B1.

Abstract LE01: DISTINGUISHED LECTURE ON TARGETS FOR CANCER PREVENTION: Prevention of cervical cancer in current and future generations by HPV vaccination and other approaches

2008-12-22

LE01

Cervical infection by a sub-set of human papillomaviruses (HPV) is the primary cause of virtually all cases of cervical cancer, which worldwide is the second most common cause of cancer deaths in women. Approximately 50% of these cancers are attributable to infection by HPV16, and another 15-20% are caused by HPV18. The remaining cases are attributable to about a dozen other HPV types. HPVs also cause a variable proportion of other malignancies, including vulvar, vaginal, penile, anal, and oropharyngeal cancer. When these cancers are associated with HPV infection, HPV16 accounts for an even higher proportion than for cervical cancer, and HPV18 is found in most of the others. The HPVs that cause these cancers are usually transmitted by sexual exposure. Genital HPV infection is extremely common, and many women are exposed to HPV soon after initiating sexual activity. It is estimated that the lifetime risk of acquiring genital HPV infection is at least a 75%. Most infections are self-limited, and regression is associated with developing a degree of type-specific resistance to reinfection. However, those infections that persist, especially with HPV16 and HPV18, place a woman at risk of cervical precancer and invasive cancer. The viral oncogenes, E6 and E7, continue to be expressed in tumors and contribute to their maintenance.

Until recently, the main approach to cervical cancer prevention has been secondary prevention by pap smear screening. However, identification of HPV as the causative agent of cervical cancer has led to two etiology-based advances. One is the use of HPV-based screening. The other is primary prevention through development of prophylactic HPV vaccines, which is the main topic of this presentation.

The current vaccines are based on the observation that the L1 main structural protein of the HPV virion can self-assemble into empty virus-like particles (VLPs) which, because they contain the conformationally-dependent neutralization epitopes of L1, can induce high levels of neutralizing antibodies. Preclinical studies showed that passive transfer of immune IgG could protect animals against high dose experimental infection, implying that neutralizing antibodies are the main mechanism for protection. Two pharmaceutical companies have developed commercial versions of the VLP vaccine. The one manufactured by GlaxoSmithKline (GSK) is a bivalent vaccine composed of VLPs from HPV16 and HPV18, while the one from Merck is a quadravalent vaccine composed of VLPs from HPV6 and HPV11 (which together account for about 90% of genital warts) in addition to VLPs from HPV16 and HPV18. The Merck vaccine is licensed in the United States (for females 9-26 years old), the European Union, and other countries, while the GSK vaccine is licensed in Europe and other countries, but has not yet been licensed in the US.

In fully immunized women, clinical efficacy trials carried out by the companies have shown that both vaccines induce almost complete protection against incident persistent genital infection attributable to the HPV types targeted by the vaccine and their associated lesions, including high-grade cervical dysplasia and, for the Merck vaccine, genital warts. Both vaccines also confer some degree of cross-protection against incident infection by closely related HPV types, but neither vaccine appears to alter the natural history of prevalent infection. The type-restricted nature of the protection implies that ~30% of potentially cancer-causing infections will not be prevented by the vaccine. Protection has been shown to last at least several years, but it remains possible that protection may wane over time, and booster vaccination could then be needed. Males have shown a similar immune response to the vaccine, but efficacy data in males have not yet been reported.

The CDC Advisory Committee on Immunization Practices (ACIP) has recommended that the main target group for the vaccine should be young adolescent girls, 11-12, because the vaccine is most cost-effective if given before sexual activity has been initiated. The ACIP has also recommended catch-up vaccination for women up to age 26. The high rate of incident infection that develops in young sexually active women implies that the vaccine is likely to prevent fewer serious infections the longer the interval between initiation of sexual activity and subsequent administration of the vaccine.

Efforts to maximize the overall reduction in cervical cancer will require coordination between vaccination and screening. A potential advantage of HPV-based screening is that its increased sensitivity compared with cytology might make it easier to safely increase the interval for screening compared with cytology. In countries with low resources, if less expensive high quality screening methods become available, lives may be saved sooner by screening and treating precancer in the current generation of women, compared with vaccination, which will be of benefit primarily to the next generation of women.

In addition to the current vaccines, it would be beneficial to develop second generation vaccines that can protect against a broader spectrum of serious HPV infections, are less expensive to produce and deliver, and require fewer doses. The companies will try to broaden protection by increasing the valency of the vaccine via the incoroporation of VLPs from other HPV types. This is a logical approach, but the manufacturing complexities could mean that such new vaccines will be expensive. In collaboration with Richard Roden at Johns Hopkins University, we are taking an alternate approach, which is to develop a vaccine based on the observation that the L2 minor capsid protein contains cross-neutralization epitopes that can induce neutralizing activity against a broad range of HPV types. Although L2 is less immunogenic than VLPs, this vaccine approach can prevent infection in preclinical models. It has not yet been tested in humans. An L2-based vaccine could be produced in bacteria and should be substantially less expensive to manufacture than VLPs.

We are also investigating the mechanisms by which HPVs infect cells and the mechanisms that underlie the efficacy of the neutralizing antibodies induced by the vaccines. To explore these issues, we have recently developed a mouse cervico-vaginal challenge model in which the mouse genital tract can be infected with what we call HPV pseudoviruses; the virions of the pseudoviruses are composed of authentic HPV capsids that, instead of encapsidating the HPV genome, have encapsidated a genome that expresses a reporter protein, which can be used as a marker of successful infection. The pseudoviruses appear to faithfully mimic the early steps of actual HPV infection. A noteworthy observation made in the mouse model is that the intact genital mucosa, including the cervix, is resistant to virion binding and, therefore, to infection. However, disruption of mucosal integrity, by gentle abrasion with a cytobrush or a detergent (nonoxynol-9), leads to the virions efficiently binding to the basement membrane, rather than to cells. Expression of the reporter protein from the pseudovirus is seen in keratinocytes 1-3 days after exposure of the genital tract to the pseudovirus. These results imply that disruption of mucosal integrity is essential for HPV infection, that the disruption enables the virus to bind to the basement membrane, and that it binds to the keratinocytes in the epithelium only after it has bound to the basement membrane. The importance of the disruption of mucosal integrity to the infectious process makes it likely that in immunized individuals, infection is prevented because there is exudation of systemic neutralizing antibodies at these potential sites of infection. This hypothesis is currently being tested.

Citation Information: Cancer Prev Res 2008;1(7 Suppl):LE01.