The Non-coding Mammary Carcinoma Susceptibility Locus, Mcs5c, Regulates Pappa Expression via Age-Specific Chromatin Folding and Allele-Dependent DNA Methylation.

In understanding the etiology of breast cancer, the contributions of both genetic and environmental risk factors are further complicated by the impact of breast developmental stage. Specifically, the time period ranging from childhood to young adulthood represents a critical developmental window in a woman's life when she is more susceptible to environmental hazards that may affect future breast cancer risk. Although the effects of environmental exposures during particular developmental Windows of Susceptibility (WOS) are well documented, the genetic mechanisms governing these interactions are largely unknown. Functional characterization of the Mammary Carcinoma Susceptibility 5c, Mcs5c, congenic rat model of breast cancer at various stages of mammary gland development was conducted to gain insight into the interplay between genetic risk factors and WOS. Using quantitative real-time PCR, chromosome conformation capture, and bisulfite pyrosequencing we have found that Mcs5c acts within the mammary gland to regulate expression of the neighboring gene Pappa during a critical mammary developmental time period in the rat, corresponding to the human young adult WOS. Pappa has been shown to positively regulate the IGF signaling pathway, which is required for proper mammary gland/breast development and is of increasing interest in breast cancer pathogenesis. Mcs5c-mediated regulation of Pappa appears to occur through age-dependent and mammary gland-specific chromatin looping, as well as genotype-dependent CpG island shore methylation. This represents, to our knowledge, the first insight into cellular mechanisms underlying the WOS phenomenon and demonstrates the influence developmental stage can have on risk locus functionality. Additionally, this work represents a novel model for further investigation into how environmental factors, together with genetic factors, modulate breast cancer risk in the context of breast developmental stage.

Mucosal associated invariant T cells from human breast ducts mediate a Th17-skewed response to bacterially exposed breast carcinoma cells.

BACKGROUND: Antimicrobial T cells play key roles in the disease progression of cancers arising in mucosal epithelial tissues, such as the colon. However, little is known about microbe-reactive T cells within human breast ducts and whether these impact breast carcinogenesis. METHODS: Epithelial ducts were isolated from primary human breast tissue samples, and the associated T lymphocytes were characterized using flow cytometric analysis. Functional assays were performed to determine T-cell cytokine secretion in response to bacterially treated human breast carcinoma cells. RESULTS: We show that human breast epithelial ducts contain mucosal associated invariant T (MAIT) cells, an innate T-cell population that recognizes specific bacterial metabolites presented by nonclassical MR1 antigen-presenting molecules. The MAIT cell population from breast ducts resembled that of peripheral blood in its innate lymphocyte phenotype (i.e., CD161, PLZF, and interleukin [IL]-18 receptor coexpression), but the breast duct MAIT cell population had a distinct T-cell receptor Vß use profile and was markedly enriched for IL-17-producing cells compared with blood MAIT cells. Breast carcinoma cells that had been exposed to Escherichia coli activated MAIT cells in an MR1-dependent manner. However, whereas phorbol 12-myristate 13-acetate/ionomycin stimulation induced the production of both interferon-γ and IL-17 by breast duct MAIT cells, bacterially exposed breast carcinoma cells elicited a strongly IL-17-biased response. Breast carcinoma cells also showed upregulated expression of natural killer group 2 member D (NKG2D) ligands compared with primary breast epithelial cells, and the NKG2D receptor contributed to MAIT cell activation by the carcinoma cells. CONCLUSIONS: These results demonstrate that MAIT cells from human breast ducts mediate a selective T-helper 17 cell response to human breast carcinoma cells that were exposed to E. coli. Thus, cues from the breast microbiome and the expression of stress-associated ligands by neoplastic breast duct epithelial cells may shape MAIT cell responses during breast carcinogenesis.

Deletion of the murine ortholog of the 8q24 gene desert has anti-cancer effects in transgenic mammary cancer models.

BACKGROUND: The gene desert on human chromosomal band 8q24 harbors multiple genetic variants associated with common cancers, including breast cancer. The locus, including the gene desert and its flanking genes, MYC, PVT1 and FAM84B, is also frequently amplified in human breast cancer. We generated a megadeletion (MD) mouse model lacking 430-Kb of sequence orthologous to the breast cancer-associated region in the gene desert. The goals were to examine the effect of the deletion on mammary cancer development and on transcript level regulation of the candidate genes within the locus. METHODS: The MD allele was engineered using the MICER system in embryonic stem cells and bred onto 3 well-characterized transgenic models for breast cancer, namely MMTV-PyVT, MMTV-neu and C3(1)-TAg. Mammary tumor growth, latency, multiplicity and metastasis were compared between homozygous MD and wild type mice carrying the transgenes. A reciprocal mammary gland transplantation assay was conducted to distinguish mammary cell-autonomous from non-mammary cell-autonomous anti-cancer effects. Gene expression analysis was done using quantitative real-time PCR. Chromatin interactions were evaluated by 3C. Gene-specific patient outcome data were analysed using the METABRIC and TCGA data sets through the cBioPortal website. RESULTS: Mice homozygous for the MD allele are viable, fertile, lactate sufficiently to nourish their pups, but maintain a 10% lower body weight mainly due to decreased adiposity. The deletion interferes with mammary tumorigenesis in mouse models for luminal and basal breast cancer. In the MMTV-PyVT model the mammary cancer-reducing effects of the allele are mammary cell-autonomous. We found organ-specific effects on transcript level regulation, with Myc and Fam84b being downregulated in mammary gland, prostate and mammary tumor samples. Through analysis using the METABRIC and TCGA datasets, we provide evidence that MYC and FAM84B are frequently co-amplified in breast cancer, but in contrast with MYC, FAM84B is frequently overexpressed in the luminal subtype, whereas MYC activity affect basal breast cancer outcomes. CONCLUSION: Deletion of a breast cancer-associated non-protein coding region affects mammary cancer development in 3 transgenic mouse models. We propose Myc as a candidate susceptibility gene, regulated by the gene desert locus, and a potential role for Fam84b in modifying breast cancer development.

The gene desert mammary carcinoma susceptibility locus Mcs1a regulates Nr2f1 modifying mammary epithelial cell differentiation and proliferation.

Genome-wide association studies have revealed that many low-penetrance breast cancer susceptibility loci are located in non-protein coding genomic regions; however, few have been characterized. In a comparative genetics approach to model such loci in a rat breast cancer model, we previously identified the mammary carcinoma susceptibility locus Mcs1a. We now localize Mcs1a to a critical interval (277 Kb) within a gene desert. Mcs1a reduces mammary carcinoma multiplicity by 50% and acts in a mammary cell-autonomous manner. We developed a megadeletion mouse model, which lacks 535 Kb of sequence containing the Mcs1a ortholog. Global gene expression analysis by RNA-seq revealed that in the mouse mammary gland, the orphan nuclear receptor gene Nr2f1/Coup-tf1 is regulated by Mcs1a. In resistant Mcs1a congenic rats, as compared with susceptible congenic control rats, we found Nr2f1 transcript levels to be elevated in mammary gland, epithelial cells, and carcinoma samples. Chromatin looping over ∼820 Kb of sequence from the Nr2f1 promoter to a strongly conserved element within the Mcs1a critical interval was identified. This element contains a 14 bp indel polymorphism that affects a human-rat-mouse conserved COUP-TF binding motif and is a functional Mcs1a candidate. In both the rat and mouse models, higher Nr2f1 transcript levels are associated with higher abundance of luminal mammary epithelial cells. In both the mouse mammary gland and a human breast cancer global gene expression data set, we found Nr2f1 transcript levels to be strongly anti-correlated to a gene cluster enriched in cell cycle-related genes. We queried 12 large publicly available human breast cancer gene expression studies and found that the median NR2F1 transcript level is consistently lower in 'triple-negative' (ER-PR-HER2-) breast cancers as compared with 'receptor-positive' breast cancers. Our data suggest that the non-protein coding locus Mcs1a regulates Nr2f1, which is a candidate modifier of differentiation, proliferation, and mammary cancer risk.

EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments.

MOTIVATION: Messenger RNA expression is important in normal development and differentiation, as well as in manifestation of disease. RNA-seq experiments allow for the identification of differentially expressed (DE) genes and their corresponding isoforms on a genome-wide scale. However, statistical methods are required to ensure that accurate identifications are made. A number of methods exist for identifying DE genes, but far fewer are available for identifying DE isoforms. When isoform DE is of interest, investigators often apply gene-level (count-based) methods directly to estimates of isoform counts. Doing so is not recommended. In short, estimating isoform expression is relatively straightforward for some groups of isoforms, but more challenging for others. This results in estimation uncertainty that varies across isoform groups. Count-based methods were not designed to accommodate this varying uncertainty, and consequently, application of them for isoform inference results in reduced power for some classes of isoforms and increased false discoveries for others. RESULTS: Taking advantage of the merits of empirical Bayesian methods, we have developed EBSeq for identifying DE isoforms in an RNA-seq experiment comparing two or more biological conditions. Results demonstrate substantially improved power and performance of EBSeq for identifying DE isoforms. EBSeq also proves to be a robust approach for identifying DE genes. AVAILABILITY AND IMPLEMENTATION: An R package containing examples and sample datasets is available at http://www.biostat.wisc.edu/kendzior/EBSEQ/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Androgen-dependent mammary carcinogenesis in rats transgenic for the Neu proto-oncogene.

Transgenic rats were created with overexpression of the Neu proto-oncogene in the mammary gland of both sexes, yet only males developed mammary cancer in an androgen-dependent fashion. Transgenic females only developed mammary cancer if treated with androgens. These tumors were positive for androgen receptor (AR), but negative for estrogen and progesterone receptors. Extensive analysis failed to detect mutations anywhere within the neu transgene from mammary carcinomas. Established mammary carcinomas eventually escaped their dependency on androgens. Transgenic long-term gonadectomized rats did not develop mammary cancer, but Neu overexpression stimulated the growth of their mammary glands. Our results suggest crosstalk between the Neu proto-oncogene and AR signaling pathways in the growth of both the normal and cancerous mammary epithelium.

The non-protein coding breast cancer susceptibility locus Mcs5a acts in a non-mammary cell-autonomous fashion through the immune system and modulates T-cell homeostasis and functions.

INTRODUCTION: Mechanisms underlying low-penetrance, common, non-protein coding variants in breast cancer risk loci are largely undefined. We showed previously that the non-protein coding mammary carcinoma susceptibility locus Mcs5a/MCS5A modulates breast cancer risk in rats and women. The Mcs5a allele from the Wistar-Kyoto (WKy) rat strain consists of two genetically interacting elements that have to be present on the same chromosome to confer mammary carcinoma resistance. We also found that the two interacting elements of the resistant allele are required for the downregulation of transcript levels of the Fbxo10 gene specifically in T-cells. Here we describe mechanisms through which Mcs5a may reduce mammary carcinoma susceptibility. METHODS: We performed mammary carcinoma multiplicity studies with three mammary carcinoma-inducing treatments, namely 7,12-dimethylbenz(a)anthracene (DMBA) and N-nitroso-N-methylurea (NMU) carcinogenesis, and mammary ductal infusion of retrovirus expressing the activated HER2/neu oncogene. We used mammary gland and bone marrow transplantation assays to assess the target tissue of Mcs5a activity. We used immunophenotyping assays on well-defined congenic rat lines carrying susceptible and resistant Mcs5a alleles to identify changes in T-cell homeostasis and function associated with resistance. RESULTS: We show that Mcs5a acts beyond the initial step of mammary epithelial cell transformation, during early cancer progression. We show that Mcs5a controls susceptibility in a non-mammary cell-autonomous manner through the immune system. The resistant Mcs5a allele was found to be associated with an overabundance of gd T-cell receptor (TCR)+ T-cells as well as a CD62L (L-selectin)-high population of all T-cell classes. In contrast to in mammary carcinoma, gdTCR+ T-cells are the predominant T-cell type in the mammary gland and were found to be overabundant in the mammary epithelium of Mcs5a resistant congenic rats. Most of them simultaneously expressed the CD4, CD8, and CD161α markers. In cultured T-cells of Mcs5a resistant congenic rats we found increased mitogen-induced proliferation and production of Th1 cytokines IFNg, IL-2, and Tumor Necrosis Factor (TNF), but not Th2 cytokines IL-4 and IL-6, or Th17 cytokine IL-17 when compared with susceptible control rats. CONCLUSIONS: These data support a hypothesis that Mcs5a displays a non-mammary cell-autonomous mechanism of action to modulate breast cancer risk through the immune system. The resistant Mcs5a allele is associated with alterations in T-cell homeostasis and functions, and overabundance of γδTCR+ T-cells in carcinogen-exposed mammary epithelium.

Intranasal administration of the chemotherapeutic perillyl alcohol results in selective delivery to the cerebrospinal fluid in rats.

Perillyl alcohol (POH) has been extensively studied for the treatment of peripheral and primary brain tumors. The intranasal route of administration has been preferred for dosing POH in early-stage clinical trials associated with promising outcomes in primary brain cancer. However, it is unclear how intranasal POH targets brain tumors in these patients. Multiple studies indicate that intranasally applied large molecules may enter the brain and cerebrospinal fluid (CSF) through direct olfactory and trigeminal nerve-associated pathways originating in the nasal mucosa that bypass the blood-brain barrier. It is unknown whether POH, a small molecule subject to extensive nasal metabolism and systemic absorption, may also undergo direct transport to brain or CSF from the nasal mucosa. Here, we compared CSF and plasma concentrations of POH and its metabolite, perillic acid (PA), following intranasal or intravascular POH application. Samples were collected over 70 min and assayed by high-performance liquid chromatography. Intranasal administration resulted in tenfold higher CSF-to-plasma ratios for POH and tenfold higher CSF levels for PA compared to equal dose intravascular administration. Our preclinical results demonstrate POH undergoes direct transport from the nasal mucosa to the CSF, a finding with potential significance for its efficacy as an intranasal chemotherapeutic for brain cancer.

Epigenetic Dysregulation of KCNK9 Imprinting and Triple-Negative Breast Cancer.

Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Here, we showed that KCNK9 is imprinted in breast tissue and identified a differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). KCNK9 hypomethylation was also found in non-cancerous tissue from 77% of women at high-risk of developing breast cancer. Functional studies demonstrated that the KCNK9 gene product, TASK3, regulates mitochondrial membrane potential and apoptosis-sensitivity. In TNBC cells and non-cancerous mammary epithelial cells from high-risk women, hypomethylation of the KCNK9 DMR predicts for increased TASK3 expression and mitochondrial membrane potential (p < 0.001). This is the first identification of the KCNK9 DMR in mammary epithelial cells and demonstration that its hypomethylation in breast cancer is associated with increases in both mitochondrial membrane potential and apoptosis resistance. The high frequency of hypomethylation of the KCNK9 DMR in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may serve as a marker of risk and a target for prevention of TNBC, particularly in African American women.

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

Freund's vaccine adjuvant promotes Her2/neu breast cancer.

BACKGROUND: Inflammation has been linked to the etiology of many organ-specific cancers. Indirect evidence suggests a possible role for inflammation in breast cancer. We investigated whether the systemic inflammation induced by Freund's adjuvant (FA) promotes mammary carcinogenesis in a rat model in which cancer is induced by the neu oncogene. METHODS: The effects of FA on hyperplastic mammary lesions and mammary carcinomas were determined in a neu-induced rat model. The inflammatory response to FA treatment was gauged by measuring acute phase serum haptoglobin. In addition, changes in cell proliferation and apoptosis following FA treatment were assessed. RESULTS: Rats receiving FA developed twice the number of mammary carcinomas as controls. Systemic inflammation following FA treatment is chronic, as shown by a doubling of the levels of the serum biomarker, haptoglobin, 15 days following initial treatment. We also show that this systemic inflammation is associated with the increased growth of hyperplastic mammary lesions. This increased growth results from a higher rate of cellular proliferation in the absence of changes in apoptosis. CONCLUSION: Our data suggests that systemic inflammation induced by Freund's adjuvant (FA) promotes mammary carcinogenesis. It will be important to determine whether adjuvants currently used in human vaccines also promote breast cancer.

Chemopreventive effects of celecoxib are limited to hormonally responsive mammary carcinomas in the neu-induced retroviral rat model.

INTRODUCTION: While current breast cancer chemoprevention strategies using selective estrogen response modulators and aromatase inhibitors are quite successful, their effects are limited to hormonally responsive breast cancer. Hormonally nonresponsive breast cancer (including estrogen receptor-negative cancer) is associated with poor prognosis for patients, and few chemoprevention agents exist for this type of cancer. The cyclooxygenase-2 inhibitor celecoxib (Celebrex) is a nonsteroidal anti-inflammatory drug and as such is a potential candidate for the prevention of hormonally nonresponsive breast cancer. METHODS: The chemopreventive effects of celecoxib were evaluated in the neu-induced retroviral rat mammary carcinogenesis model, to assess the efficacy of celecoxib on hormonally responsive and hormonally nonresponsive mammary carcinomas. RESULTS: Dietary celecoxib at 1,200 mg/kg diet was highly efficacious in the prevention of hormonally responsive mammary carcinomas in intact rats, decreasing tumor multiplicity by 56% (P < 0.0001) and by 74% (P = 0.0002) in two independent experiments. No significant effect was found, however, on hormonally nonresponsive mammary carcinomas of ovariectomized rats. Treatment with a combination diet, consisting of tamoxifen at 2 mg/kg diet and celecoxib at 1,200 mg/kg diet, reduced tumor multiplicity by 72% (P = 0.0002) in intact rats. This reduction was not statistically different from that observed with celecoxib alone. Furthermore, long-term treatment with celecoxib was not associated with reductions in tumor volume in either intact rats or ovariectomized rats. In contrast, tamoxifen treatment and the combination regimen caused significant reductions in tumor volumes in intact rats (P = 0.01 and P = 0.004, respectively). Consistent with these data, decreases in proliferation and increases in apoptosis were detected in tamoxifen-treated and combination diet-treated tumors. No such modulations were observed in celecoxib-treated tumors. CONCLUSION: The chemopreventive effects of celecoxib appear to be limited to modulations in multiplicity of hormonally responsive mammary carcinomas. The fact that no synergistic or additive effects were observed in combination diet-treated rats raises the question of whether celecoxib is suitable for the prevention of hormonally nonresponsive breast cancer or for use in combination therapy with selective estrogen response modulators or aromatase inhibitors.

Neu-induced retroviral rat mammary carcinogenesis: a novel chemoprevention model for both hormonally responsive and nonresponsive mammary carcinomas.

Clinically relevant animal models of mammary carcinogenesis are crucial for the development and evaluation of new breast cancer chemopreventive agents. The neu-induced retroviral rat mammary carcinogenesis model is based on the direct in situ transfer of the activated neu oncogene into the mammary epithelium using a replication-defective retroviral vector. The resulting mammary carcinomas in intact Wistar-Furth rats exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas can be prevented by tamoxifen treatment. In ovariectomized animals, the mammary carcinomas are hormonally nonresponsive and cannot be prevented by tamoxifen. We evaluated the efficacy of retinoic X receptor-selective retinoids (rexinoids) in this novel model of mammary carcinogenesis. The rexinoids LG100268 and bexarotene (LG1069, Targretin) were highly efficacious in the prevention of neu-induced mammary carcinomas. Dietary LG100268 at 100 mg/kg diet decreased tumor multiplicity by 32% (P = 0.0114) in intact rats and 50% (P < 0.0001) in ovariectomized rats. Bexarotene treatment at a dose of 250 mg/kg diet was associated with reductions in tumor multiplicity of 84% (P < 0.0001) and 86% (P < 0.0001) in intact and ovariectomized animals, respectively. In addition to tumor multiplicity, proliferation and apoptosis were modulated by bexarotene treatment independently of estrogen signaling. The neu-induced retroviral rat mammary carcinogenesis model represents a valuable addition to existing rodent chemoprevention models. The model is useful for assessing the efficacy of chemopreventive agents, specifically those compounds that target hormonally nonresponsive tumors.

Genetically engineered rat models for breast cancer.

Rat mammary cancers generally resemble many features of human breast cancer. With the recent developments in rat genetic engineering, the rat has become an excellent model system to study aspects of the molecular etiology of breast cancer. In this review, we describe the efforts to generate genetically engineered rat models for breast cancer.

Production of knockout rats using ENU mutagenesis and a yeast-based screening assay.

The rat is a widely used model in biomedical research and is often the preferred rodent model in many areas of physiological and pathobiological research. Although many genetic tools are available for the rat, methods to produce gene-disrupted knockout rats are greatly needed. In this study, we developed protocols for creating N-ethyl-N-nitrosourea (ENU)-induced germline mutations in several rat strains. F1 preweanling pups from mutagenized Sprague Dawley (SD) male rats were then screened for functional mutations in Brca1 and Brca2 using a yeast gap-repair, ADE2-reporter truncation assay. We produced knockout rats for each of these two breast cancer suppressor genes.

Fine mapping reveals multiple loci and a possible epistatic interaction within the mammary carcinoma susceptibility quantitative trait locus, Mcs5.

To identify high-frequency, low-penetrance breast cancer modifier genes, we have developed a rat genetic model that uses the Wistar-Kyoto (WKy) inbred strain, resistant to developing 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis, as a congenic donor and the susceptible Wistar-Furth (WF) strain as the recipient. Here, data from congenic rat lines containing smaller WKy genomic intervals of the Mcs5 quantitative trait locus region are presented to fine map three independently acting Mcs5 subloci. WKy-homozygous females from congenic lines defining Mcs5a, Mcs5b, and Mcs5c averaged, respectively, 4.0 +/- 0.4, 11.6 +/- 0.6, and 3.5 +/- 0.4 mammary carcinomas per rat. These phenotypic values are statistically different from the WF-homozygous phenotype value of 8.0 +/- 0.4, which is the baseline phenotype used for these experiments. We identified a likely Mcs5a x Mcs5b epistatic interaction that results in masking the increased susceptibility effect of the Mcs5b WKy allele by the Mcs5a WKy allele. We also provide evidence for a Mcs5a x Mcs5c interaction that is synergistic to decrease mammary carcinoma susceptibility below the additive effects of WKy alleles at each locus independently. The Mcs5 subloci are currently localized to 1.0, 7.5, and 4.5 Mb of rat chromosome 5, and the orthologous regions are on human chromosome 9 and mouse chromosome 4. These loci will provide unbiased candidate gene loci for evaluation in human case-control association studies.

Perillyl alcohol inhibits a calcium-dependent constitutive nuclear factor-kappaB pathway.

The cell death induced by the monoterpene anticancer agent perillyl alcohol correlates to the increased expression of certain proapoptotic genes known to influence cell survival. Whereas sequence-specific DNA-binding factors dictate the expression patterns of genes through transcriptional regulation, those transcriptional factors influencing constitutive cell survival with perillyl alcohol treatment are not well studied. Here, we investigated whether the monoterpenes can regulate the activity of nuclear factor-kappaB (NF-kappaB), a calcium-dependent transcription factor necessary for survival in the WEHI-231 B-lymphoma cells. Unique among the monoterpenes, perillyl alcohol short-term treatment induced a persistent decrease of calcium levels, whereas other various monoterpenes caused transient reductions in calcium levels. Perillyl alcohol treatment also rapidly elicited reductions of NF-kappaB DNA-binding activity and target gene induction, which was associated with an increase in apoptosis in these B-lymphoma cells. This apoptosis was directly due to NF-kappaB because its prior activation abolished the cell killing effects of perillyl alcohol treatment. Our findings suggest that perillyl alcohol can inhibit NF-kappaB function to modulate gene expression patterns and cell survival of certain B-lymphoma cells. The effects of perillyl alcohol were not limited to these B-lymphoma cells but were also observed in MDA-MB 468 cells, an estrogen receptor-negative breast cancer cell line. These results identify a calcium-dependent NF-kappaB pathway as a molecular target of perillyl alcohol activity in different cancer cell types.

Regions of H- and K-ras that provide organ specificity/potency in mammary cancer induction.

Organ-specific cancers with activated ras oncogenes most often are associated exclusively with only one ras isoform. For example, only H-ras activation is associated with rat mammary cancers. The mechanism underlying this specificity is mostly unknown. We have shown previously that this tissue specificity of Ras isoforms is defined by the Ras protein itself and not by differential gene expression among Ras family members. Here we show that elements in the known domains in the hypervariable region of Ras (amino acids 170-189) interact in part to control this mammary/H-Ras specificity. In addition, these in vivo mammary studies for the first time identify domains in the mostly homologous region of Ras (amino acids 1-169) that strongly influence the oncogenic potency/specificity of H-Ras.

Congenic rats reveal three independent Copenhagen alleles within the Mcs1 quantitative trait locus that confer resistance to mammary cancer.

It has previously been shown that the Copenhagen (COP) rat contains several genetic loci that contribute to its mammary tumor-resistant phenotype after 7,12-dimethylbenz(a)anthracene (DMBA) administration. One of these loci, mammary carcinoma susceptibility 1 (Mcs1), is located on the centromeric end of chromosome 2 and appears to act in a semidominant fashion. To confirm the existence and independent action of this locus and also aid in the identification of the physical location of the Mcs1 gene, congenic lines were generated by transferring the Mcs1 COP allele onto a Wistar Furth (WF) genetic background. Male carriers were genotyped using microsatellite markers spanning 20-30 cM of the Mcs1 locus. One of the congenic lines minimally retained the COP allele at D2Mit29 on the centromeric end of chromosome 2 and extended distally to D2Rat201. Heterozygous Mcs1 carrier rats were interbred, and the female offspring were treated with DMBA. The female rats from the Mcs1 congenic line that carried one or two COP alleles of the Mcs1 region had a significantly reduced (65 and 85%, respectively) tumor development (P < 0.001) compared with rats carrying zero COP alleles at this locus. A WF.COP-D2Mit29/D2Rat201 homozygous congenic strain derived at the N10 generation was treated with DMBA, and the COP homozygous rats developed 1.5 +/- 0.3 carcinomas/rat versus 6.3 +/- 0.5 in WF control rats (P < 0.0001). Fine mapping of this congenic interval using several recombinant lines identified three genetic loci within the Mcs1 congenic region that independently supported a tumor resistance phenotype. These genetic loci have been termed Mcs1a, Mcs1b, and Mcs1c. In rats for which each locus was homozygous for the COP allele, tumor development was reduced by approximately 60% compared with littermate controls. The identification of these independent loci within the Mcs1 COP allele provide a model of the genetic complexity of cancer.