Minshu Yu

Mammary gland carcinogenesis by food-derived heterocyclic amines and studies on the mechanisms of carcinogenesis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

The heterocyclic amines (HCAs) comprise a family of mutagenic/carcinogenic compounds found in cooked meat. Several HCAs including 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are mammary gland carcinogens in rats. One mammary gland carcinogen, PhIP, is the most prevalent in the human diet. This article reviews the mechanisms of mammary gland carcinogenesis of PhIP including metabolic processing, DNA adduct formation, effects on mammary gland development, cell signaling, and the genomic alterations found in PhIP-induced rat mammary gland carcinomas.

Id4 Regulates Mammary Epithelial Cell Growth and Differentiation and Is Overexpressed in Rat Mammary Gland Carcinomas

Id4 belongs to a family of helix-loop-helix (HLH) proteins that impact cellular growth and differentiation via regulation of basic HLH transcription factors. Herein the rat Id4 gene was cloned (GenBank Accession No. AF468681). The expression of rat Id4 was examined in rat mammary gland tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in the human diet. By real-time polymerase chain reaction analysis, relative expression of Id4 mRNA in carcinomas, adenomas, and normal tissue was 27, 6, and 1, respectively. Immunohistochemical analysis indicated statistically elevated nuclear expression for Id4 protein in carcinomas in comparison to adenomas and normal mammary gland. In carcinomas, Id4 nuclear expression was positively correlated with proliferation, invasiveness, and tumor weight (Fisher Exact Test or Spearman Correlation, P < 0.05). The consequence of enforced expression of Id4 on mammary epithelial cell proliferation, differentiation, and growth in soft agar was examined in HC11 cells, a well-characterized model for studying various aspects of mammary epithelial cell biology. After transient and stable transfection of HC11 cells, Id4 overexpression increased cell proliferation and inhibited lactogenic hormone-mediated differentiation as revealed by inhibition of beta-casein promoter activity and beta-casein expression. In addition, enforced expression of Id4 in HC11 cells induced a statistically significant increase in colony growth in soft agar. The results implicate Id4 in rat mammary gland carcinogenesis and suggest that Id4 may contribute to carcinogenesis by inhibiting mammary epithelial cell differentiation and stimulating mammary epithelial cell growth.

Possible role of Stat5a in rat mammary gland carcinogenesis

Signal transducer and activator of transcription (Stat) 5a is a transcription factor mediating the action of specific cytokines, growth factors and hormones on gene expression. In the mammary gland, Stat5a is well recognized for its function in prolactin signaling, lobuloalveolar development, and milk protein expression during pregnancy and lactation. Latent cytoplasmic Stat5a is activated by tyrosine phosphorylation and following dimerization undergoes nuclear import. In the current study, Stat5a expression was examined immunohistochemically in carcinomas induced by the chemical carcinogens 7,12-dimethylbenz[a]anthracene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. A high percentage of carcinomas showed nuclear labeling of Stat5a [44 of 68 (65%)] with Stat5a nuclear labeling index ranging from 18 to 77%. In contrast, control normal mammary gland tissue displayed cytosolic expression. Carcinomas with different Stat5a staining patterns (cytoplasmic or nuclear) showed a statistical difference for the proliferating cell nuclear antigen (PCNA) labeling, tumor differentiation, nuclear grade, mitotic activity, and tumor size. High Stat5a nuclear expression was closely correlated with the higher-grade carcinomas. Stat5a nuclear expression was also detected in intraductal proliferations (10 of 21 lesions) and in ductal carcinomas in situ (13 of 15 lesions). Immunohistochemical analysis was further carried out in human breast cancers. Stat5a nuclear expression was detected in ductal and lobular carcinomas and DCIS at a frequency of 48% (15/31), 33% (2/6), and 40% (2/5), respectively. Nuclear expression of Stat5a in human breast cancers also correlated with the PCNA nuclear labeling index. The findings implicate activated Stat5a in mammary gland cancer development in the rat and human.

Genomic imbalance in rat mammary gland carcinomas induced by 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine

2‐Amino‐1‐methyl‐6‐phenylimidazo(4,5‐b)pyridine (PhIP), a compound found in cooked meat, is a mammary gland carcinogen in female Sprague‐Dawley rats. PhIP‐induced rat mammary gland carcinomas were examined for mutations in several genes (exons) known to regulate cell growth and apoptosis, including p53 (4–8), p21Waf1 (coding region), Apc (14, 15), B‐catenin (3), E‐cadherin (9,13,15), Bcl‐x (coding region), Bax (3), IGFIIR (28), and TGFBIIR (3). DNA from 30 carcinomas was examined by single‐strand conformation polymorphism analysis, but no mutations were detected in these genes or gene regions. DNA from carcinomas and matching normal tissue were further screened for allelic imbalance by using a polymerase chain reaction–based approach with primers to known microsatellite regions located throughout the rat genome. Of 53 markers examined, 12 revealed allelic imbalance. Microsatellite instability (MSI) was detected at two markers, one on chromosome 4 and one on chromosome 6. Sixty‐five percent and 96% of all carcinomas examined (N=23) showed MSI at these loci on chromosomes 4 and 6, respectively, supporting the notion that MSI plays a role in PhIP‐induced mammary carcinogenesis. Loss of heterozygosity (LOH), an indication of a possible tumor suppressor gene, was observed at 10 markers distributed on chromosomes 3, 10, 11, 14, and X. The frequency of LOH at these markers was 75–94%, supporting that the regions of allelic imbalance were largely similar for the PhIP‐induced carcinomas examined in this study. When PhIP‐induced carcinomas from rats placed on high‐fat and low‐fat diet were compared, no unique regions of allelic imbalance or statistical differences in the frequency of allelic imbalance were observed. Therefore, the high‐fat diet, known to be a promoter of PhIP‐induced rat mammary carcinogenesis, did not appear to influence allelic imbalance in the carcinomas. Interestingly, 7,12‐dimethylbenz[a]‐anthracene–induced mammary carcinomas did not show allelic imbalance at 11 of the 12 loci that showed allelic imbalance in PhIP‐induced carcinomas. These findings suggest that distinct chemical carcinogens induce different patterns of allelic imbalance during rat mammary carcinogenesis. Since several of the known genes involved in carcinogenesis did not harbor mutations in PhIP‐induced carcinomas, further studies are needed to clarify the critical genes involved in PhIP‐induced mammary carcinogenesis and to determine whether regions of LOH harbor potentially novel tumor suppressor genes involved in this disease. Mol. Carcinog. 27:76–83, 2000.

Gene expression profiling in the mammary gland of rats treated with 7,12‐dimethylbenz[a]anthracene

Identification of molecular markers of early‐stage breast cancer development is important for the diagnosis and prevention of the disease. In the present study, we used microarray analysis to examine the differential expression of genes in the rat mammary gland soon after treatment with a known chemical carcinogen, 7,12‐dimethylbenz[a]anthracene (DMBA), and prior to tumor development. Six weeks after DMBA, differential expression of multiple genes involved in cell growth, differentiation and microtubule dynamics were observed. Gene expression changes were further validated by a combination of techniques, including real‐time PCR, RT‐PCR, Western blotting and immunohistochemistry. An inhibition of differentiation in this early stage was suggested by the lower expression of β‐casein and transferrin and higher expression of hsp27 in glands from DMBA‐treated rats. Possible cell cycle deregulation was indicated by an increased expression of cyclin D1 and hsp86, a heat shock protein associated with cyclin D1. Prior to tumor development, DMBA increased cellular proliferation as detected by Ki‐67 and stathmin immunostaining in histologically normal mammary gland. Genes regulating microtubule function, including stathmin, Ran, α‐tubulin and hsp27, were all overexpressed in the mammary gland of DMBA‐treated rats, raising the possibility that disruption of microtubule dynamics and abnormal mitosis may be critical events preceding breast cancer development. Several of the altered proteins, including hsp27, hsp86 and stathmin, may ultimately serve as markers of early breast cancer development. Published 2005 Wiley‐Liss, Inc.

Susceptibility of Rats to Mammary Gland Carcinogenesis by the Food-Derived Carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine (PhIP) Varies with Age and Is Associated with the Induction of Differential Gene Expression

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, induces mammary gland cancer when administered to adolescent female rats (43-day-old). In contrast, mature virgin rats (150-day-old) were resistant to mammary carcinogenesis by PhIP. To explore the possible mechanisms for the age-related differences in susceptibility, PhIP-DNA adduct levels, mutations, and gene expression were examined in glands from 43-day and 150-day-old PhIP-treated rats. In rats of different ages, PhIP-DNA adduct levels detected by the (32)P-post-labeling assay and mutant frequency measured in the lacI reporter gene of Big Blue rats were not statistically different. PhIP-DNA adduct levels, adduct removal, and mutation burden did not appear to account for the variation in carcinogen susceptibility with age. However, cDNA microarray analysis indicated that PhIP treatment differentially altered the profile of gene expression in glands from 43-day-old and 150-day-old rats. In 150-day-old rats, PhIP enhanced the expression of genes associated with differentiation (eg, beta-casein, kappa-casein, whey acidic protein) and induced morphological differentiation. In contrast, in 43-day-old rats, PhIP inhibited the expression of differentiation genes and enhanced cellular proliferation. From 3 hours to 6 weeks after PhIP dosing, the number of clones showing altered expression declined more than 50% in 150-day-old rats but increased fourfold in 43-day-old rats (29 clones versus 194, respectively) suggesting that PhIP induced a cascade of gene expression alterations only in susceptible rats. Genes showing altered expression specifically in 43-day-old rats included the Ras superfamily genes and genes associated with protein synthesis/degradation (lysosomal proteins, heat shock proteins, and proteasomes). The microarray data support the notion that the mechanism of age-dependent susceptibility to mammary gland cancer is largely associated with differential responses in expression of genes involved in cellular differentiation, proliferation, and protein homeostasis.

Global Gene Expression Profiling of Chemically Induced Rat Mammary Gland Carcinomas and Adenomas

Chemical carcinogens induce both benign and malignant mammary gland tumors in female Sprague-Dawley rats. To identify gene expression profiles associated with malignancy, cDNA microarray analysis was used to compare gene expression profiles in rat mammary gland carcinomas, adenomas, and normal mammary gland. Tumors were induced with various chemical carcinogens including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), 7-12-dimethylbenz[a]anthracene (DMBA), N-nitrosomethylurea (NMU), and 4-aminobiphenyl. The global gene expression profiles in carcinomas and adenomas were distinguishable by hierarchical clustering and multi-dimensional scaling analyses. Permutation analysis revealed 110 clones statistically differentially expressed between benign and malignant tumors (p < 0.0005). Carcinomas showed relatively high expression of several genes associated with mammary epithelial cell growth and proliferation (e.g., cyclin D1, PDGFα) and relatively low expression of differentiation marker genes (e.g., β-casein, whey acidic protein, transferrin). Other categories of genes showing differential expression between carcinomas and adenomas were associated with protein homeostasis, cytoskeleton, extracellular matrix, and cell metabolism (fatty acid metabolism, oxidative phosphorylation, and glycolysis). Major gene families implicated in malignancy by over-expression in carcinomas included the annexins (annexin A1 and A4) and Stat family of transcription factors (Stat3 and Stat5a). The elevated expression of the prolactin receptor in carcinomas concomitant with several components of the mitogenic prolactin signaling pathway implicated prolactin/prolactin receptor/Stat5a/cyclin D1 in rat mammary gland malignancy.

Allelic imbalance and altered expression of genes in chromosome 2q11-2q16 from rat mammary gland carcinomas induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a compound found in cooked meat, is a mammary gland carcinogen in rats. Comparative genomic hybridization of PhIP-induced rat mammary gland carcinomas revealed loss in the centromeric region of 2q, a region known to carry the mammary carcinoma susceptibility 1 (Mcs1) gene and several other genes relevant to carcinogenesis. Allelic imbalance, specifically microsatellite instability and loss of heterozygosity, was examined in mammary gland carcinomas induced by PhIP in Sprague–Dawley (SD)×Wistar Furth F1 hybrid rats. In a polymerase chain reaction (PCR)-based assay with 34 microsatellite markers coinciding to 2q11–2q16, nine markers revealed allelic imbalance. The frequency of imbalance in the tumors varied from 10 to 100% depending on the specific marker. However, none of the markers coinciding with the Mcs1 gene locus showed allelic imbalance, suggesting that alterations at this locus were not associated with PhIP-induced rat mammary gland cancer. The expression of several genes physically mapped to 2q11–2q16 and potentially involved in carcinogenesis including Ccnb (cyclin B1), Ccnh (cyclin H), Rasa (Ras GAP), Rasgrf2, Pi3kr1 (p85α), and Il6st (gp130) was also examined by quantitative real-time PCR and immunohistochemistry (IHC) across a large bank of PhIP-induced SD rat mammary gland carcinomas. By quantitative real-time PCR, the mRNA expression of Rasa, Pi3kr1, Ccnh, and Il6st in carcinomas was, respectively, 22-, 20-, three- and threefold higher in carcinomas than in control mammary gland tissues (P<0.05, Students t-test). A statistically sixfold lower expression of Rasgrf2 was detected in carcinomas whereas no significant change in Ccnb1 expression was observed. The findings from quantitative real-time PCR were confirmed by IHC for each gene. In addition, the proliferation index in mammary gland carcinomas as assessed by PCNA was found to correlate with the overexpression of Cyclin H by IHC analysis (P<0.05, Spearman Rank Order Correlation). The findings from the current study implicate molecular alterations in the proximal region of 2q in PhIP-induced rat mammary gland carcinomas.