Differential preventive activity of sulindac and atorvastatin in Apc+/Min-FCCCmice with or without colorectal adenomas.

OBJECTIVE: The response of subjects to preventive intervention is heterogeneous. The goal of this study was to determine if the efficacy of a chemopreventive agent differs in non-tumour-bearing animals versus those with colorectal tumours. Sulindac and/or atorvastatin was administered to Apc+/Min-FCCC mice with known tumour-bearing status at treatment initiation. DESIGN: Male mice (6-8 weeks old) underwent colonoscopy and received control chow or chow with sulindac (300 ppm), atorvastatin (100 ppm) or sulindac/atorvastatin. Tissues were collected from mice treated for 14 weeks (histopathology) or 7 days (gene expression). Cell cycle analyses were performed on SW480 colon carcinoma cells treated with sulindac, atorvastatin or both. RESULTS: The multiplicity of colorectal adenomas in untreated mice bearing tumours at baseline was 3.6-fold higher than that of mice that were tumour free at baseline (P=0.002). Atorvastatin completely inhibited the formation of microadenomas in mice that were tumour free at baseline (P=0.018) and altered the expression of genes associated with stem/progenitor cells. Treatment of tumour-bearing mice with sulindac/atorvastatin led to a 43% reduction in the multiplicity of colorectal adenomas versus untreated tumour-bearing mice (P=0.049). Sulindac/atorvastatin increased the expression of Hoxb13 and Rprm significantly, suggesting the importance of cell cycle regulation in tumour inhibition. Treatment of SW480 cells with sulindac/atorvastatin led to cell cycle arrest (G0/G1). CONCLUSIONS: The tumour status of animals at treatment initiation dictates response to therapeutic intervention. Atorvastatin eliminated microadenomas in tumour-free mice. The tumour inhibition observed with Sul/Atorva in tumour-bearing mice was greater than that achieved with each agent.

Estrogen metabolism within the lung and its modulation by tobacco smoke.

Although estrogen and the enzymes responsible for its metabolism have been detected within the lung, the ability of this tissue to metabolize estrogen has not been demonstrated previously. The goal of this study was to characterize the profile of estrogen metabolites within the murine lung and to determine the effect of tobacco smoke exposure on metabolite levels. Use of liquid chromatography-tandem mass spectrometry led to the detection of three estrogens (E1, E2 and E3) and five estrogen metabolites (2-OHE1, 4-OHE1, 4-OHE2, 2-OMeE1 and 2-OMeE2) within the perfused lung, with 4-OHE1 being the most abundant species. Levels of 4-OHEs, carcinogenic derivatives produced primarily by cytochrome P450 1B1 (Cyp1b1), were 2-fold higher in females than males. Deletion of Cyp1b1 in females led to a dramatic reduction (21-fold) in 4-OHEs, whereas levels of 2-OHE1 and the putative protective estrogen metabolite 2-OMeE2 were increased (2.4- and 5.0-fold, respectively) (P = 0.01). Similar quantitative differences in estrogen metabolite levels were observed between Cyp1b1 null and wild-type males. Exposure of female mice to tobacco smoke for 8 weeks (2h per day, 5 days per week) increased the levels of 4-OHE1 (4-fold) and 2-OHE2 (2-fold) within the lung while reducing the total concentration of 2-OMeEs to 70% of those of unexposed controls. These data suggest that tobacco smoke accelerates the production of 4-OHEs within the lung; carcinogenic metabolites that could potentially contribute to lung tumor development. Thus, inhibition of CYP1B1 may represent a promising strategy for the prevention and treatment of lung cancer.

High Variability in Cellular Proliferation, Gene Expression, and Cytokine Production in the Nonneoplastic Colonic Epithelium of Young Apc+/Min-FCCC Mice.

An urgent need exists to identify efficacious therapeutic preventive interventions for individuals who are at high risk of developing colorectal cancer. To maximize the benefits of preventive intervention, it is vital to identify the time interval during which the initiation of a preventive intervention will lead to an optimal outcome. The goal of the present study was to determine if oncogenic events can be detected in the nonneoplastic colonic mucosa of Apc+/Min-FCCC mice prior to formation of the first adenoma, thus defining an earlier point of intervention along the cancer continuum. Tissues taken at three potential points of intervention were characterized: prior to Apc mutation (wild type Apc+/+-FCCC mice); after initiation but prior to colon adenoma formation (tumor-free Apc+/Min-FCCC mice); and after formation of the first colon adenoma (tumor-bearing Apc+/Min-FCCC mice). Experimentation focused on molecular processes that are dysregulated in early colon lesions: 1) cellular proliferation (proliferative index and size of the proliferative zone); 2) cellular stemness (expression of Ascl2, Grem1, Lgr5 and Muc2); 3) EGFR signaling (expression of Ereg); and 4) inflammation (expression of Mmp9, Ptsg2, and Reg4, as well as secretion of 18 cytokines involved in immune activation and response). Interestingly, the nonneoplastic colonic mucosa of wild type, tumor-free Apc+/Min-FCCC , and tumor-bearing Apc+/Min-FCCC mice did not display significant differences in average epithelial cell proliferation (fold change 0.8-1.3, p≥0.11), mucosal gene expression (fold change 0.8-1.4, p≥0.22), or secretion of specific cytokines from colonic mucosa (fold change 0.2-1.5, p≥0.06). However, the level of cytokine secretion was highly variable, with many (22% of wild type, 31% of tumor-free Apc+/Min-FCCC , and 31% of tumor-bearing Apc+/Min-FCCC ) mice categorized as outliers (> 1.5 x interquartile ranges below the first quartile or above the third quartile) due to elevated expression of at least one cytokine. In summary, no differences were observed in proliferation, stemness, and EGFR signaling in the colonic mucosa of wild type vs Apc+/Min-FCCC mice, with low baseline cytokine expression, prior to the formation of the first colon adenoma. The results of this study provide valuable baseline data to inform the design of future cancer prevention studies.

Novel Protein-Based Vaccine against Self-Antigen Reduces the Formation of Sporadic Colon Adenomas in Mice.

Novel immunopreventive strategies are emerging that show great promise for conferring long-term protection to individuals at high risk of developing colorectal cancer. The KISIMA vaccine platform utilizes a chimeric protein comprising: (1) a selected tumor antigen; (2) a cell-penetrating peptide to improve antigen delivery and epitope presentation, and (3) a TLR2/4 agonist to serve as a self-adjuvant. This study examines the ability of a KISIMA vaccine against achaete-scute family bHLH transcription factor 2 (Ascl2), an early colon cancer antigen, to reduce colon tumor formation by stimulating an anti-tumor immune response. Vaccine administrations were well-tolerated and led to circulating antibodies and antigen-specific T cells in a mouse model of colorectal cancer. To assess preventive efficacy, the vaccine was administered to mice either alone or in combination with the immune checkpoint inhibitor anti-PD-1. When delivered to animals prior to colon tumor formation, the combination strategy significantly reduced the development of colon microadenomas and adenomas, as compared to vehicle-treated controls. This response was accompanied by an increase in the intraepithelial density of CD3+ T lymphocytes. Together, these data indicate that the KISIMA-Ascl2 vaccine shows great potential to be a safe and potent immunopreventive intervention for individuals at high risk of developing colorectal cancer.

Allelic imbalance in BRCA1 and BRCA2 gene expression is associated with an increased breast cancer risk.

The contribution of BRCA1 and BRCA2 to familial and non-familial forms of breast cancer has been difficult to accurately estimate because of the myriad of potential genetic and epigenetic mechanisms that can ultimately influence their expression and involvement in cellular activities. As one of these potential mechanisms, we investigated whether allelic imbalance (AI) of BRCA1 or BRCA2 expression was associated with an increased risk of developing breast cancer. By developing a quantitative approach utilizing allele-specific real-time PCR, we first evaluated AI caused by nonsense-mediated mRNA decay in patients with frameshift mutations in BRCA1 and BRCA2. We next measured AI for BRCA1 and BRCA2 in lymphocytes from three groups: familial breast cancer patients, non-familial breast cancer patients and age-matched cancer-free females. The AI ratios of BRCA1, but not BRCA2, in the lymphocytes from familial breast cancer patients were found to be significantly increased as compared to cancer-free women (BRCA1: 0.424 versus 0.211, P = 0.00001; BRCA2: 0.206 versus 0.172, P = 0.38). Similarly, the AI ratios were greater for BRCA1 and BRCA2 in the lymphocytes of non-familial breast cancer cases versus controls (BRCA1: 0.353, P = 0.002; BRCA2: 0.267, P = 0.03). Furthermore, the distribution of under-expressed alleles between cancer-free controls and familial cases was significantly different for both BRCA1 and BRCA2 gene expression (P < 0.02 and P < 0.02, respectively). In conclusion, we have found that AI affecting BRCA1 and to a lesser extent BRCA2 may contribute to both familial and non-familial forms of breast cancer.

High density DNA array analysis reveals distinct genomic profiles in a subset of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) generally harbor activating mutations in KIT or platelet-derived growth facter receptor (PDGFRA). Mutations in these receptor tyrosine kinases lead to dysregulation of downstream signaling pathways that contribute to GIST pathogenesis. GISTs with KIT or PDGFRA mutations also undergo secondary cytogenetic alterations that may indicate the involvement of additional genes important in tumor progression. Approximately 10-15% of adult and 85% of pediatric GISTs do not have mutations in KIT or in PDGFRA. Most mutant adult GISTs display large-scale genomic alterations, but little is known about the mutation-negative tumors. Using genome-wide DNA arrays, we investigated genomic imbalances in a set of 31 GISTs, including 10 KIT/PDGFRA mutation-negative tumors from nine adults and one pediatric case and 21 mutant tumors. Although all 21 mutant GISTs exhibited multiple copy number aberrations, notably losses, eight of the 10 KIT/PDGFRA mutation-negative GISTs exhibited few or no genomic alterations. One KIT/PDGFRA mutation-negative tumor exhibiting numerous genomic changes was found to harbor an alternate activating mutation, in the serine-threonine kinase BRAF. The only other mutation-negative GIST with significant chromosomal imbalances was a recurrent metastatic tumor found to harbor a homozygous deletion in chromosome arm 9p. Similar findings in several KIT-mutant GISTs identified a minimal overlapping region of deletion of approximately 0.28 Mbp in 9p21.3 that includes only the CDKN2A/2B genes, which encode inhibitors of cell-cycle kinases. These results suggest that GISTs without activating kinase mutations, whether pediatric or adult, generally exhibit a much lower level of cytogenetic progression than that observed in mutant GISTs.

Lyophilized açaí pulp (Euterpe oleracea Mart) attenuates colitis-associated colon carcinogenesis while its main anthocyanin has the potential to affect the motility of colon cancer cells.

This study evaluated the possible protective effects of lyophilized açaí pulp (AP) in a colitis-associated carcinogenesis (CAC) rat model and the modifying effect of cyanidin 3-rutinoside (C3R) on the motility of RKO colon adenocarcinoma cells, using the wound healing assay. Male Wistar rats were induced to develop CAC using 1,2-dimethylhydrazine (DMH) and 2,4,6-trinitrobenzene acid (TNBS). Animals were randomly assigned to different groups that received basal diet or basal diet supplemented with 5.0% or 7.5% lyophilized AP. The findings indicate: 1) C3R (25 µM) has the potential to reduce RKO cell motility in vitro; 2) ingestion of lyophilized AP reduces the total number of aberrant crypt foci (ACF), ACF multiplicity, tumor cell proliferation and incidence of tumors with high grade dysplasia; 3) AP increases the gene expression of negative regulators of cell proliferation such as Dlc1 and Akt3, as well as inflammation (Ppara). Thus, lyophilized AP could exert a potential antitumor activity.

Haploinsufficiency in tumor predisposition syndromes: altered genomic transcription in morphologically normal cells heterozygous for VHL or TSC mutation.

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal "one-hit" cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the "Warburg effect". Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NFκB and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.