Folic Acid Supplementation Promotes Hypomethylation in Both the Inflamed Colonic Mucosa and Colitis-Associated Dysplasia.

PURPOSE: The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. METHODS: Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). RESULTS: A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/ß-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. CONCLUSIONS: High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

Dysregulation of miR-1-3p: An Early Event in Colitis-Associated Dysplasia.

Detection of colorectal dysplasia during surveillance colonoscopy remains the best method of determining risk for colitis-associated colorectal cancer (CAC). miRNAs (miRs) show great promise as tissue-specific biomarkers of neoplasia. The goal of this study was to explore the miR expression profile of precancerous dysplastic lesions in the AOM/DSS mouse model and identify early molecular changes associated with CAC. Epithelial cells were laser-microdissected from the colonic mucosa (inflamed versus dysplastic) of mice with AOM/DSS-induced colitis. A miR signature that can distinguish inflamed non-neoplastic mucosa from dysplasia was identified. Bioinformatic analyses led to the discovery of associated miR gene targets and enriched pathways and supported the construction of a network interaction map. miR-1a-3p was one of the miRs with the highest number of predicted targets, including Cdk6. Interestingly, miR-1a-3p and Cdk6 were down- and up-regulated in dysplastic lesions, respectively. Transfection of HCT116 and RKO cells with miR-1a-3p mimics induced apoptosis and cell cycle arrest in G1, suggesting its biological function. A slight reduction in the level of CDK6 transcripts was also observed in cells transfected with miR-1. These data provide novel insight into the early molecular alterations that accompany the development of CAC and identify a miR signature that represents a promising biomarker for the early detection of colitis-associated dysplasia.

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.

Screening of Health-Associated Oral Bacteria for Anticancer Properties in vitro.

While extensive literature exists about the role of oral bacterial pathogens like Porphyromonas gingivalis and Fusobacterium nucleatum in oral squamous cell carcinoma (OSCC), the role of health-associated species has been largely unexplored. In this study, we assessed the effect of Streptococcus mitis, Rothia mucilaginosa, Neisseria flavescens, Haemophilus parainfluenzae, Lautropia mirabilis, and Veillonella parvula on proliferation and expression of marker genes (IL-6, TNF-α, MMP3, CD36, CCD1, and NANOG) in OSCC cell lines CAL27, SCC25, and SCC4. Porphyromonas gingivalis was included as a pathogenic control. Both bacterial lysates (3 concentrations) and live cells (3 MOIs) were tested. S. mitis, H. parainfluenzae, and N. flavescens resulted in substantial, dose-dependent reduction of proliferation, which was found to be mediated by H2O2 for the former and intracellular infection in the latter two species. However, only H. parainfluenzae showed differential antiproliferative effect against the cancer cell lines vs. the normal control (TIGKs). In the gene expression assays, the health-associated species mostly downregulated CD36, a gene that plays an important role in tumor growth and metastasis, while P. gingivalis upregulated it. IL6 and TNF expression, on the other hand, was upregulated by almost all species, particularly the Gram-negatives including P. gingivalis. The effect on other genes was less evident and varied significantly by cell line. This exploratory study is the first insight into how health-associated bacteria may interact with OSCC. Further studies to explore whether the observed effects may have implications for the prevention or treatment of oral cancer are warranted.

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.

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.

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.

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.

Altered gene expression in morphologically normal epithelial cells from heterozygous carriers of BRCA1 or BRCA2 mutations.

We hypothesized that cells bearing a single inherited "hit" in a tumor suppressor gene express an altered mRNA repertoire that may identify targets for measures that could delay or even prevent progression to carcinoma. We report here on the transcriptomes of primary breast and ovarian epithelial cells cultured from BRCA1 and BRCA2 mutation carriers and controls. Our comparison analyses identified multiple changes in gene expression, in both tissues for both mutations, which were validated independently by real-time reverse transcription-PCR analysis. Several of the differentially expressed genes had been previously proposed as cancer markers, including mammaglobin in breast cancer and serum amyloid in ovarian cancer. These findings show that heterozygosity for a mutant tumor suppressor gene can alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner; these detectable effects of "one hit" represent early molecular changes in tumorigenesis that may serve as novel biomarkers of cancer risk and as targets for chemoprevention.

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.

Molecular mechanisms of action of imatinib mesylate in human ovarian cancer: a proteomic analysis.

BACKGROUND: Imatinib mesylate (Gleevec, Novartis, Basel, Switzerland) is a small-molecule tyrosine kinase inhibitor with activity against ABL, BCR-ABL, c-KIT, and PDGFR alpha. Several clinical trials have evaluated the efficacy and safety of imatinib in patients with ovarian carcinoma who have persistent or recurrent disease following front-line platinum/taxane based chemotherapy. However, there is limited pre-clinical and clinical data on the molecular targets and action of imatinib in ovarian cancer. MATERIALS AND METHODS: Human ovarian cancer cells (A2780) were treated with imatinib mesylate for either 6 or 24 h. We employed a 2D (two-dimensional) gel electrophoresis and mass spectrometry-based proteomics approach to identify protein expression patterns and signaling pathways that were altered in response to imatinib. Cells were analyzed for PDGFR alpha and AKT expression, which were then correlated with imatinib sensitivity. RESULTS: Using 2D gel electrophoresis of overlapping pH ranges from pH 4 to 11, about 4,000 protein spots could be analyzed reproducibly. Proteins whose levels changed between twofold to 30 fold were grouped according to whether changes were in the same direction at both time points of treatment with respect to the control, or changed their levels only at one of the time points. CONCLUSION: Differentially regulated proteins following imatinib treatment of A2780 cells involved the regulation of actin cytoskeleton, metabolic pathways, cell cycle, cell proliferation, apoptosis, cell junctions, and signal transduction. Thus, exposure of cells to imatinib produces complex changes in the cell that require further investigation.

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.

Prominent expression of metalloproteinases in early stages of ovarian tumorigenesis.

The role for matrix metalloproteinases (MMPs) in tumor cells invasion and metastasis is well established, and expression of MMPs is recognized as an indication of tumor cell malignancy. Previous studies suggest that the degradation of the basement membrane is a crucial early step in epithelial transformation and ovarian tumorigenesis. Thus, MMPs may also express and exert a role in preneoplastic lesions of ovarian tissues. We investigated the expression of the major metalloproteinases, gelatinase A, 72 kDa type IV collagenase (MMP-2), and gelatinase B, 92 kDa type IV collagenase (MMP-9), and the presence of basement membrane in ovarian tumors and tissues from prophylactic oophorectomies using immunostaining. MMP expression was also characterized in a panel of ovarian cancer cell lines and several nontumorigenic ovarian surface epithelial primary cells by zymography, Northern, and Western blots. We found, surprisingly, that MMP-2 and MMP-9 are expressed more frequently in early lesions than in established carcinomas. No correlation was found between the expression of MMPs and tumor grades or stages. In preneoplastic lesions, MMP-2 or MMP-9 expression often associates with the absence of basement membrane and morphological alterations. MMP-2 is often expressed in nontumorigenic ovarian surface epithelial cells but reduced or absent in cancer cells. Thus, we conclude that MMPs expression does not correlate with the malignancy of ovarian epithelial cells as generally thought. Rather, increased metalloproteinase expression is an early event in ovarian tumorigenesis and associates with the loss of epithelial basement membrane and morphological transformation. We propose that the increased MMP activity is an etiological factor for ovarian cancer risk. We found that MMPs expression does not correlate with the malignancy of ovarian epithelial cells as generally thought. Rather, increased metalloproteinase expression is an early event in ovarian tumorigenesis. The finding suggests roles of MMP in tumor initiation in addition to invasion, and may impact on the strategy for use of MMP inhibitors in cancer prevention.

Perception of differentiation cues by GATA factors in primitive endoderm lineage determination of mouse embryonic stem cells.

The formation of the primitive endoderm covering the inner cell mass of early mouse embryos can be simulated in vitro by the differentiation of mouse embryonic stem (ES) cells in culture following either aggregation of suspended cells or stimulation of cell monolayers with retinoic acid. The developmentally regulated transcription factors GATA-4 and GATA-6 have determining role in mouse extraembryonic endoderm development. We analyzed the in vitro differentiation of mouse embryonic stem cells deficient of GATA factors and conclude that GATA-4 is required for ES cells to perceive a cell positioning (cell aggregation) signal and GATA-6 is required to sense morphogenic (retinoic acid) signal. The collaboration between GATA-6 and GATA-4, or GATA-6 and GATA-5 which can substitute for GATA-4, is involved in the perception of differentiation cues by embryonic stem cells in their determination of endoderm lineage. This study indicates that the lineage differentiation of ES cells can be manipulated by the expression of GATA factors.

Anomalous expression of epithelial differentiation-determining GATA factors in ovarian tumorigenesis.

Tumor cells often appear in a deviant differentiated stage, and dedifferentiation is a hallmark of malignancy; however, the causative mechanism of the global changes in dedifferentiation is not understood. The GATA transcription factors function in cell lineage specification during embryonic development and organ formation. The transcriptional targets of the GATA factors in early embryonic development include Disabled-2 and collagen IV, markers for epithelial lineages. GATA-4 and GATA-6 are expressed strongly and are localized in the nucleus in ovarian surface epithelial cells in tissues or primary cell cultures. By immunohistochemistry, we found that 82% of the 50 tumors analyzed had lost GATA-6 function, either by a complete absence of expression or by cytoplasmic mislocalization. The frequent loss of GATA-6 was also confirmed in a panel of ovarian surface epithelial and tumor cell lines. Although GATA-4 is absent only in a small percentage (14%) of ovarian tumors, it is lost in the majority of established cell lines in culture. The loss of GATA-6 correlates with the loss of Disabled-2, collagen IV, and laminin, markers for epithelial cell types. Loss of GATA factors was also found in an in vitro model for spontaneous transformation of rat ovarian epithelial cells. Repression of GATA-6 by small interfering (si)RNA approach in cultured cells leads to dedifferentiation as indicated by the loss of Disabled-2 and laminin expression. Restoration of GATA factors expression by ectopic transfection suppresses cell growth and is incompatible with the maintenance of the cells in culture. However, restoration of GATA-4 and GATA-6 expression is not able to induce expression of endogenous Disabled-2 in tumor cells, suggesting that the loss of GATA factors and dedifferentiation are irreversible processes. In conclusion, we observed the inappropriate expression and cellular localization of the GATA transcription factors in ovarian tumor tissues and cancer cell lines, and we have demonstrated that down-regulation of GATA factor expression leads to dedifferentiation. We propose that alterations of GATA transcription factor expression and aberrant nucleocytoplasmic localization may contribute to the anomalous epithelial dedifferentiation of the ovarian tumor cells.

Hypomethylation of the synuclein gamma gene CpG island promotes its aberrant expression in breast carcinoma and ovarian carcinoma.

Recent studies indicate that synuclein gamma (SNCG) gene, located in chromosome 10, participates in the pathogenesis of the breast and ovary. SNCG, also known as breast cancer-specific gene 1 (BCSG1), is not expressed in normal mammary or ovarian surface epithelial cells but is highly expressed in the vast majority of advanced staged breast and ovarian carcinomas. When overexpressed, SNCG significantly stimulates breast cancer proliferation and metastasis. To fully understand the molecular mechanisms underlying the abnormal expression of SNCG in neoplastic diseases, in this study, we extensively examined the methylation status of a CpG island located in exon 1 of SNCG gene in a panel of breast and ovarian tumor-derived cell lines to determine whether DNA methylation plays a crucial role in SNCG expression. In vivo bisulfite DNA sequencing of genomic DNA isolated from breast cancer cell lines showed that the 15 CpG sites within the CpG island were completely unmethylated in all SNCG-positive cell lines (5 of 5), but were densely and heterogeneously methylated in the majority of SNCG-negative cell lines (3 of 4). The methylation occurred primarily at the CpG sites 2, 5, 7, and 10-15. Similarly, we observed a strong correlation of hypomethylation of the CpG island and SNCG expression in ovarian cancer cell lines (5 of 5). Intriguingly, the methylation pattern in ovarian cancer cells is different from that in breast cancer cells. In SNCG-nonexpressing ovarian cancer cells, all 15 of the CpG sites were completely methylated instead of selective methylation at certain sites shown in breast cancer cells, thereby suggesting a tissue-specific methylation pattern. A correlation between hypomethylation of the exon 1 and expression of SNCG mRNA was also observed in primary breast tumor tissues. The importance of DNA methylation in the control of SNCG expression in cancer cells is further strengthened by demonstration of re-expression of SNCG mRNA in SNCG-negative ovarian and breast cancer cells with a demethylating agent 5-aza-2'-deoxycytidine. In addition, we demonstrate that inhibition of cell growth leads to a decreased mRNA expression and an increased DNA methylation of SNCG gene. Taken together, these new findings strongly suggest that DNA hypomethylation is a common mechanism underlying the abnormal expression of this candidate oncogene in breast and ovarian carcinomas.

Dynamic alterations of the extracellular environment of ovarian surface epithelial cells in premalignant transformation, tumorigenicity, and metastasis.

BACKGROUND: Ovarian surface epithelial cells are positionally organized as a single cell layer by a sheet of basement membrane. It is believed that the contact of the ovarian surface epithelial cells with the basement membrane regulates cell growth and ensures the organization of the epithelium. Disabled-2 (Dab2), a signal transduction protein and a candidate tumor suppressor of ovarian carcinoma, functions in positional organization of ovarian surface epithelial cells. In ovarian carcinomas, genetic and epigenetic changes enable the tumor cells to escape positional control and proliferate in a disorganized fashion. Alterations in the extracellular environment may also be critical for tumor initiation and progression. METHODS: We analyzed and compared the presence of collagen IV and laminin, the scaffold proteins of the basement membrane, and Dab2 in 50 ovarian tumors that are restricted to the ovaries and in 50 metastases of ovarian tumors by immunohistochemistry. Expression of collagen IV, laminin, and Dab2 was also analyzed by Northern blotting in a panel of human ovarian surface epithelial and cancer cell lines. RESULTS: The basement membrane is often absent in morphologically benign ovarian surface and cyst epithelium and low-grade tumors and collagen IV and laminin are absent in the extracellular matrix of most of the primary tumors tested. Of the 50 ovarian tumors confined to the ovaries, 6% (3 of 50) were collagen IV positive and 24% (12 of 50) were laminin positive tumors. Of the 50 metastatic tumors, 16% (8 of 50) are collagen IV positive and 86% (43 of 50) are laminin positive. In addition, even in the metastatic ovarian tumors that are largely collagen IV negative, there are pockets of local areas in which the tumor cells are surrounded by collagen IV-positive staining. Dab2 is absent in the majority of ovarian tumors found in both ovaries and metastatic sites. In both nontumorigenic human ovarian surface epithelial and cancer cell lines, collagen IV, laminin, and Dab2 are expressed aberrantly. CONCLUSIONS: Loss of the basement membrane may be an early event in the preneoplastic transformation of ovarian surface epithelium and in the early stages of tumorigenesis before tumor invasion and metastasis. The majority of primary ovarian tumors examined lack collagen IV and laminin in their extracellular matrix. However, expression of laminin is restored in the majority of metastatic tumors. Reexpression of collagen IV may also contribute to tumor metastasis. The ability of tumor cells to dynamically alter the expression of collagen IV and laminin may facilitate the shedding of cancer cells into the peritoneal spaces and subsequent attachment to the metastatic sites. We propose that loss of collagen IV and laminin may be an initial event in ovarian tumorigenicity and that restoration of collagen IV and laminin expression in the later stages of tumor development may promote metastasis of ovarian tumors.

DNA array-based method for detection of large rearrangements in the BRCA1 gene.

In most families with multiple cases of breast and ovarian cancer, the cancer appears to be associated with germline alterations in BRCA1 or BRCA2. However, somatic mutations in BRCA1 and BRCA2 in sporadic breast and ovarian tumors are rare, even though loss of heterozygosity in BRCA1 and BRCA2 loci in these tumors appears frequently. This may be attributed to mutation detection assays that detect alterations in the coding regions and splice site junctions, but that miss large gene rearrangements. To look specifically for mutations such as large gene rearrangements that span several kilobases (kb) of genomic DNA, we have developed a fluorescence DNA microarray assay. This assay rapidly and simultaneously screens for such rearrangements along the entire gene. In our screen of 15 malignant ovarian tumors, we found one sample with a novel 3-kb deletion encompassing exon 17 of BRCA1 that leads to a frameshift mutation. This deletion was not detected in the corresponding constitutive DNA. Our results indicate that, whereas somatic mutations in BRCA1 appear to be rare in ovarian cancers, the search for large gene rearrangements should be included in any BRCA1 mutational analysis. Furthermore, the method described in this report has the potential to screen clinical tumor samples for genomic rearrangements simultaneously in a large number of cancer-associated genes.

OVCA2 is downregulated and degraded during retinoid-induced apoptosis.

Retinoids, the natural and synthetic derivatives of vitamin A, have been shown to regulate the growth and differentiation of a wide variety of cell types and consequently have enormous potential as chemotherapeutic agents. We have previously identified 2 genes, termed OVCA1 and OVCA2, which are located in a small region showing a high frequency of allelic loss in breast and ovarian tumors and share a common exon. Recent studies have suggested that expression of OVCA1 may be influenced by retinoids. Therefore, we analyzed the expression of OVCA1 and OVCA2 in cells in response to treatment with all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR), or under conditions of low serum and confluence, to determine further the roles of OVCA1 and OVCA2 in cell growth, apoptosis and differentiation. We show that OVCA2 mRNA and protein are ubiquitously expressed and that they are downregulated in the lung cancer cell line Calu-6 after treatment with RA and 4HPR. In addition, we observed that OVCA2 protein is proteolytically degraded in response to RA and 4HPR treatment in a time- and dose-dependent manner in the promyelocytic leukemia cell line HL60. In contrast, expression of the candidate tumor suppressor OVCA1 was not downregulated by these treatments. Furthermore, we demonstrate that OVCA2 is evolutionarily conserved and shows regional homology with dihydrofolate reductases (DHFRs), specifically with hydrolase folds found in alpha-beta hydrolases. Our results are in contrast to a previous report and show that OVCA2, not OVCA1 mRNA and protein, is downregulated in response to RA and 4HPR.