Rat Mammary carcinoma susceptibility 3 (Mcs3) pleiotropy, socioenvironmental interaction, and comparative genomics with orthologous human 15q25.1-25.2.

Genome-wide association studies of breast cancer susceptibility have revealed risk-associated genetic variants and nominated candidate genes; however, the identification of causal variants and genes is often undetermined by genome-wide association studies. Comparative genomics, utilizing Rattus norvegicus strains differing in susceptibility to mammary tumor development, is a complimentary approach to identify breast cancer susceptibility genes. Mammary carcinoma susceptibility 3 (Mcs3) is a Copenhagen (COP/NHsd) allele that confers resistance to mammary carcinomas when introgressed into a mammary carcinoma susceptible Wistar Furth (WF/NHsd) genome. Here, Mcs3 was positionally mapped to a 7.2-Mb region of RNO1 spanning rs8149408 to rs107402736 (chr1:143700228-150929594, build 6.0/rn6) using WF.COP congenic strains and 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis. Male and female WF.COP-Mcs3 rats had significantly lower body mass compared to the Wistar Furth strain. The effect on female body mass was observed only when females were raised in the absence of males indicating a socioenvironmental interaction. Furthermore, female WF.COP-Mcs3 rats, raised in the absence of males, did not develop enhanced lobuloalveolar morphologies compared to those observed in the Wistar Furth strain. Human 15q25.1-25.2 was determined to be orthologous to rat Mcs3 (chr15:80005820-82285404 and chr15:83134545-84130720, build GRCh38/hg38). A public database search of 15q25.1-25.2 revealed genome-wide significant and nominally significant associations for body mass traits and breast cancer risk. These results support the existence of a breast cancer risk-associated allele at human 15q25.1-25.2 and warrant ultrafine mapping of rat Mcs3 and human 15q25.1-25.2 to discover novel causal genes and variants.

Evaluation, Acceptance, and Qualification of Digital Measures: From Proof of Concept to Endpoint.

To support the successful adoption of digital measures into internal decision making and evidence generation for medical product development, we present a unified lexicon to aid communication throughout this process, and highlight key concepts including the critical role of participant engagement in development of digital measures. We detail the steps of bringing a successful proof of concept to scale, focusing on key decisions in the development of a new digital measure: asking the right question, optimized approaches to evaluating new measures, and whether and how to pursue qualification or acceptance. Building on the V3 framework for establishing verification and analytical and clinical validation, we discuss strategic and practical considerations for collecting this evidence, illustrated with concrete examples of trailblazing digital measures in the field.

A Method to Pre-Screen Rat Mammary Gland Whole-Mounts Prior To RNAscope.

RNAscope is a quantitative in situ gene expression measurement technique that preserves the spatial aspect of intact tissue; thus, allowing for comparison of specific cell populations and morphologies. Reliable and accurate measurement of gene expression in tissue is dependent on preserving RNA integrity and the quantitative nature of RNAscope. The purpose of this study was to determine if the quantitative nature of RNAscope was retained following processing and carmine staining of mammary gland whole-mounts, which are commonly used to identify lesions, such as hyperplasia and ductal carcinoma in situ (DCIS). We were concerned that handling and procedures required to visualize microscopic disease lesions might compromise RNA integrity and the robustness of RNAscope. No effect on the quantitative abilities of RNAscope was detected when mammary gland whole-mounts were pre-screened for lesions of interest prior to RNAscope. This was determined in comparison to tissue that had been formalin-fixed and paraffin embedded (FFPE) immediately after collection. The ability to pre-screen whole-mounts allowed unpalpable diseased lesions to be identified without labor-intensive serial sectioning of tissue samples to find diseased tissue. This method is applicable to evaluate mammary gland whole-mounts during normal mammary gland development, function, and disease progression.

Chemical carcinogen-induced rat mammary carcinogenesis is a potential model of p21-activated kinase positive female breast cancer.

The p21-activated kinase 1 (PAK1) gene encodes a serine/threonine kinase that is overexpressed in a subset of human breast carcinomas with poor prognosis. The laboratory rat (Rattus norvegicus) orthologous gene is located at Mammary carcinoma susceptibility 3 (Mcs3) QTL on rat chromosome 1. We used quantitative PCR to determine effects of Mcs3 genotype and 7,12-dimethylbenz(a)anthracene (DMBA) exposure on Pak1 expression. There was no effect of Mcs3 genotype; however, there was a 3.5-fold higher Pak1 level in DMBA-exposed mammary glands (MGs) than in unexposed glands (P < 0.05). Sequence variants in Pak1 exons did not alter amino acid sequence between Mcs3-susceptible and -resistant strains. Protein expression of PAK1/Pak1 in human breast carcinomas and DMBA-exposed rat mammary glands was detected using immunohistochemistry (IHC). Rat mammary glands from 12-wk-old females unexposed to DMBA were negative for Pak1, whereas 24% of carcinogen-exposed mammary glands from age-matched females stained positive for Pak1. The positive mammary glands exposed to carcinogen had no pathological signs of disease. Human breast carcinomas, used as comparative controls, had a 22% positivity rats. This was consistent with other human breast cancer studies of PAK1 expression. Similar frequencies of human/rat PAK1/Pak1 expression in female breast carcinomas and carcinogen-induced rat mammary glands, showing no visible pathogenesis of disease, suggests aberrant PAK1 expression is an early event in development of some breast cancers. Laboratory rats will be a useful experimental organism for comparative studies of Pak1-mediated mechanisms of breast carcinogenesis. Future studies of PAK1 as a diagnostic marker of early breast disease are warranted.

Benefits of Case-Based versus Traditional Lecture-Based Instruction in a Preclinical Removable Prosthodontics Course.

This study compared the acceptability and relative effectiveness of case-based learning (CBL) versus traditional lecture-based (LB) instruction in a preclinical removable prosthodontics course in the University of North Carolina at Chapel Hill School of Dentistry DDS curriculum. The entire second-year class (N=82) comprised this crossover study's sample. Assessments of baseline comprehension and confidence in removable partial denture (RPD) treatment planning were conducted at the beginning of the course. Near the end of the course, half of the class received CBL and LB instruction in an RPD module in alternating sequence, with students serving as their own control group. Assessments of perceived RPD treatment planning efficacy, comprehension, and instruction method preference were administered directly after students completed the RPD module and six months later. Analyses of variance accounting for period, carryover, and sequence effects were used to determine the relative effects of each approach using a p<0.05 statistical significance threshold. The results showed that the students preferred CBL (81%) over LB instruction (9%), a pattern that remained unchanged after a six-month period. Despite notable period and carryover effects, CBL was also associated with higher gains in RPD treatment planning comprehension (p=0.04) and perceived efficacy (p=0.01) compared to LB instruction. These gains diminished six months after the course-a finding based on a 49% follow-up response rate. Overall, the students overwhelmingly preferred CBL to LB instruction, and the findings suggest small albeit measurable educational benefits associated with CBL. This study's findings support the introduction and further testing of CBL in the preclinical dental curriculum, in anticipation of possible future benefits evident during clinical training.

Congenic rats with higher arylamine N-acetyltransferase 2 activity exhibit greater carcinogen-induced mammary tumor susceptibility independent of carcinogen metabolism.

BACKGROUND: Recent investigations suggest role(s) of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer. Rat NAT2 is orthologous to human NAT1 and the gene products are functional homologs. We conducted in vivo studies using F344.WKY-Nat2 rapid/slow rats, congenic at rat Nat2 for high (rapid) and low (slow) arylamine N-acetyltransferase activity, to assess a possible role for rat NAT2 in mammary tumor susceptibility. METHODS: Mammary carcinogens, methylnitrosourea (MNU) and 7,12-dimethylbenzanthracene (DMBA) neither of which is metabolized by N-acetyltransferase, were administered to assess mammary tumors. MNU was administered at 3 or 8 weeks of age. DMBA was administered at 8 weeks of age. NAT2 enzymatic activity and endogenous acetyl-coenzyme A (AcCoA) levels were measured in tissue samples and embryonic fibroblasts isolated from the congenic rats. RESULTS: Tumor latency was shorter in rapid NAT2 rats compared to slow NAT2 rats, with statistical significance for MNU administered at 3 and 8 weeks of age (p = 0.009 and 0.050, respectively). Tumor multiplicity and incidence were higher in rapid NAT2 rats compared to slow NAT2 rats administered MNU or DMBA at 8 weeks of age (MNU, p = 0.050 and 0.035; DMBA, p = 0.004 and 0.027, respectively). Recombinant rat rapid-NAT2, as well as tissue samples and embryonic fibroblasts derived from rapid NAT2 rats, catalyzed p-aminobenzoic acid N-acetyl transfer and folate-dependent acetyl-coenzyme A (AcCoA) hydrolysis at higher rates than those derived from rat slow-NAT2. Embryonic fibroblasts isolated from rapid NAT2 rats displayed lower levels of cellular AcCoA than slow NAT2 rats (p < 0.01). CONCLUSIONS: A novel role for rat NAT2 in mammary cancer was discovered unrelated to carcinogen metabolism, suggesting a role for human NAT1 in breast cancer.

Physical Confirmation and Comparative Genomics of the Rat Mammary carcinoma susceptibility 3 Quantitative Trait Locus.

Human breast and rat mammary cancer susceptibility are complex phenotypes where complete sets of risk associated loci remain to be identified for both species. We tested multiple congenic rat strains to physically confirm and positionally map rat Mammary carcinoma susceptibility 3 (Mcs3)-a mammary cancer resistance allele previously predicted at Rattus norvegicus chromosome 1 (RNO1). The mammary cancer susceptible Wistar Furth (WF) strain was the recipient, and the mammary cancer resistant Copenhagen (Cop) strain was the RNO1-segment donor for congenics. Inbred WF females averaged 6.3 carcinogen-induced mammary carcinomas per rat. Two WF.Cop congenic strains averaged 2.8 and 3.4 mammary carcinomas per rat, which confirmed Mcs3 as an independently acting allele. Two other WF.Cop congenic strains averaged 6.6 and 8.1 mammary carcinomas per rat, and, thus, did not contain Mcs3 Rat Mcs3 was delimited to 27.8 Mb of RNO1 from rs8149408 to rs105131702 (RNO1:143700228-171517317 of RGSC 6.0/rn6). Human genetic variants with p values for association to breast cancer risk below 10-7 had not been reported for Mcs3 orthologous loci; however, human variants located in Mcs3-orthologous regions with potential association to risk (10-7 < p < 10-3) were listed in some population-based studies. Further, rat Mcs3 contains sequence orthologous to human 11q13/14-a region frequently amplified in female breast cancer. We conclude that Mcs3 is an independently acting mammary carcinoma resistance allele. Human population-based, genome-targeted association studies interrogating Mcs3 orthologous loci may yield novel breast cancer risk associated variants and genes.

Out-of-pocket medical costs and third-party healthcare costs for children with Down syndrome.

Prior analyses have estimated the lifetime total societal costs of a person with Down syndrome (DS); however, no studies capture the expected medical costs that patients with DS can expect to incur during childhood. The study utilized the OptumHealth Reporting and Insights administrative claims database from 1999 to 2013. Children with a diagnosis of DS were identified, and their time was divided into clinically relevant age categories. Patients with DS in each age category were matched to controls without chromosomal conditions. Out-of-pocket medical costs and third-party expenditures were compared between the patient-age cohorts with DS and matched controls. Patients with DS had significantly higher mean annual out-of-pocket costs than their matched controls within each age and cost category. Total annual incremental out-of-pocket costs associated with DS were highest among individuals from birth to age 1 ($1,907, P < 0.001). The main drivers of the incremental out-of-pocket costs associated with DS were inpatient costs in the 1st year of life ($925, P < 0.001) and outpatient costs in later years (ranging $183-$623, all P < 0.001). Overall, patients with DS incurred incremental out-of-pocket medical costs of $18,248 between birth and age 18 years; third-party payers incurred incremental costs of $230,043 during the same period. Across all age categories, mean total out-of-pocket annual costs were greater for individuals with DS than those of matched controls. On average, parents of children with DS pay an additional $84 per month for out-of-pocket medical expenses when costs are amortized over 18 years. © 2016 Wiley Periodicals, Inc.

Identification and functional analysis of acute myeloid leukemia susceptibility associated single nucleotide polymorphisms at non-protein coding regions of RUNX1.

Little is known about the susceptibility to acute myeloid leukemia. We aim to search non-protein coding regions of key hematopoiesis transcription factors for genetic variations associated with acute myeloid leukemia susceptibility. We genotyped SNPs of RUNX1 P1 promoter, P2 promoter, +23 enhancer, intron 5.2 enhancer, PU.1 promoter, CEBPA promoter, and CEBPE promoter from acute myeloid leukemia patients and healthy controls. Rs2249650 and rs2268276 at RUNX1 intron 5.2 enhancer were found to be associated with acute myeloid leukemia susceptibility. Artificial reporters containing different rs2249650 and rs2268276 alleles showed differential activities in the K562 cell line, a human immortalized myeloid leukemia line. Rs2249650 contributes to reporter activities more than rs2268276. Gel shift assay is consistent with the luciferase assay. Supershift assay indicated that one potential binding protein was PU.1. To sum up, rs2268276 and especially rs2249650 may be qualified as new acute myeloid leukemia susceptibility-associated SNPs.

Differential 12-O-Tetradecanoylphorbol-13-acetate-induced activation of rat mammary carcinoma susceptibility Fbxo10 variant promoters via a PKC-AP1 pathway.

Rat mammary carcinoma susceptibility 5a1 (Mcs5a1), which is concordant to human MCS5A1 breast cancer risk locus, mediates susceptibility by a non-mammary cell-autonomous mechanism associated with T cell differential expression of F-box protein 10 (Fbxo10). Human FBXO10, an evolutionarily conserved ubiquitin ligase gene, was shown to have a potential role in regulating cell death by controlling the degradation of Bcl-2, a key protein involved in apoptosis. Breast cancer susceptibility is controlled by interactions between environmental and genetic factors; therefore, we sought to determine if breast cancer risk-associated environmental chemicals interact with Mcs5a1 variants using luciferase reporter constructs containing 4.2 kb Fbxo10 promoters based on alleles of mammary cancer susceptible Wistar Furth (WF) and resistant Wistar Kyoto (WKY) rat strains. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced activation of a 4.2 kb WF Fbxo10 promoter region, but lower levels of activation of the homologous WKY Fbxo10 promoter region. Using general and specific protein kinase inhibitors, we identified a protein kinase C (PKC) pathway that mediated TPA activation. We narrowed the possible PKCs to a member of the atypical PKC isoforms, namely PKCµ. We also determined that activator protein 1 (AP1) family member c-Fos mediated TPA activation of the 4.2 kb WF Fbxo10 promoter. TPA was shown to induce endogenous FBXO10 mRNA and FBXO10 protein in Jurkat cells, a human T cell line, with a maximal level of expression from 1.5 to 2.5 h after exposure. These results indicate that FBXO10/Fbxo10 expression is regulated by a PKC-dependent pathway acting through c-Fos, which binds AP1-specific DNA elements in Mcs5a1.

Identifying Motor, Emotional-Behavioral, and Cognitive Deficits that Comprise the Triad of HD Symptoms from Patient, Caregiver, and Provider Perspectives.

BACKGROUND: The objective of this study was to identify important attributes associated with the triad of symptoms (cognition, emotional-behavioral, and motor) of Huntington's disease (HD) from patient, caregiver, and medical provider perspectives to facilitate development of a new disease-specific, health-related quality of life (HRQOL) instrument. METHODS: We conducted a targeted literature review of HD and HRQOL instruments, expert surveys, and patient and caregiver phone-based interviews to extract information on the symptoms and issues most relevant to the HD symptom triad (HD triad). The data collected from these sources were used to generate themes and subdomains and to develop an integrated schema that highlights the key dimensions of the triad. RESULTS: THE SEARCH IDENTIFIED THE FOLLOWING AREAS: emotional functioning/behavioral changes (e.g., positive emotions, sadness/depression); cognitive functioning (e.g., memory/learning, attention/comprehension); physical functioning (e.g., motor functioning, medication); social functioning (e.g., leisure, interpersonal relationships); end-of-life concerns/planning; and gene testing. Fifteen individuals diagnosed with HD and 16 HD caregivers, recruited from several Huntington's Disease Society of America support group networks, completed phone interviews. Nineteen US medical providers who specialize in HD completed the online survey. Twenty-six subdomains of the HD symptom triad (seven cognition, 12 emotional-behavioral, and seven motor) emerged relatively consistently across patient, caregiver, and provider samples. These included movements/chorea, memory impairment, depression, and anxiety. DISCUSSION: Based on an integrated, mixed-methods approach, important HD triad symptom were identified and organized into a guiding schema. These patient-, caregiver-, and provider-triangulated data served as the basis for development of a HD-specific HRQOL instrument, the HD-PRO-TRIAD™.

HD-PRO-TRIAD™ Validation: A Patient-reported Instrument for the Symptom Triad of Huntington's Disease.

BACKGROUND: Few valid, disease-specific measures of health-related quality of life (HRQOL) capture the spectrum of symptoms associated with Huntington's disease (HD). The HD-PRO-TRIAD™ is a new, HD-specific, patient-reported outcome (PRO) instrument of the HD symptom triad (cognitive decline, emotional/behavioral dyscontrol, and motor dysfunction) designed for clinical research and practice. The objective was to validate the HD-PRO-TRIAD™ through a cross-sectional sample of individuals with HD and caregivers. METHODS: Development of the HD-PRO-TRIAD™ has been described elsewhere. A total of 132 individuals with HD and 40 HD caregivers, comprising 29 dyads, participated in the cross-sectional psychometric validation of this instrument. Participants provided responses to the HD-PRO-TRIAD™ and other HRQOL and disease severity instruments (EuroQOL 5D, Short Form 12, Neuro-QOL Item Banks, PROMIS Global Health, and self-reported Unified Huntington's Disease Rating Scale Total Functional Capacity and Independence Scales). Internal consistency, construct validity, and patient-caregiver proxy consistency were evaluated. RESULTS: Internal consistency of the three domains and overall HD-PRO-TRIAD™ instrument was supported by Cronbach's alpha values ≥0.94. Construct validity was supported by significant correlations between HD-PRO-TRIAD™ domain scores and other measures of the same domains (e.g., significant positive correlations between HD-PRO-TRIAD™ Anxiety with Neuro-QOL Anxiety), as well as slightly weaker but still strong correlations with other HRQOL instruments (e.g., HD-PRO-TRIAD™ Anxiety and UHDRS Independence; all p<0.01). Consistency between patient self-report and caregiver proxy report was supported by an intra-class correlation coefficient ≥0.92 for all three domains and the overall instrument. DISCUSSION: These data indicate that HD-PRO-TRIAD™ is a reliable and valid HRQOL instrument that captures the typical triad of HD symptoms.

Significant overlap between human genome-wide association-study nominated breast cancer risk alleles and rat mammary cancer susceptibility loci.

INTRODUCTION: Human population-based genome-wide association (GWA) studies identify low penetrance breast cancer risk alleles; however, GWA studies alone do not definitively determine causative genes or mechanisms. Stringent genome- wide statistical significance level requirements, set to avoid false-positive associations, yield many false-negative associations. Laboratory rats (Rattus norvegicus) are useful to study many aspects of breast cancer, including genetic susceptibility. Several rat mammary cancer associated loci have been identified using genetic linkage and congenic strain based-approaches. Here, we sought to determine the amount of overlap between GWA study nominated human breast and rat mammary cancer susceptibility loci. METHODS: We queried published GWA studies to identify two groups of SNPs, one that reached genome-wide significance and one comprised of SNPs failing a validation step and not reaching genome- wide significance. Human genome locations of these SNPs were compared to known rat mammary carcinoma susceptibility loci to determine if risk alleles existed in both species. Rat genome regions not known to associate with mammary cancer risk were randomly selected as control regions. RESULTS: Significantly more human breast cancer risk GWA study nominated SNPs mapped at orthologs of rat mammary cancer loci than to regions not known to contain rat mammary cancer loci. The rat genome was useful to predict associations that had met human genome-wide significance criteria and weaker associations that had not. CONCLUSIONS: Integration of human and rat comparative genomics may be useful to parse out false-negative associations in GWA studies of breast cancer risk.

Human MCS5A1 candidate breast cancer susceptibility gene FBXO10 is induced by cellular stress and correlated with lens epithelium-derived growth factor (LEDGF).

Genetic variation and candidate genes associated with breast cancer susceptibility have been identified. Identifying molecular interactions between associated genetic variation and cellular proteins may help to better understand environmental risk. Human MCS5A1 breast cancer susceptibility associated SNP rs7042509 is located in F-box protein 10 (FBXO10). An orthologous Rattus norvegicus DNA-sequence that contains SNV ss262858675 is located in rat Mcs5a1, which is part of a mammary carcinoma susceptibility locus controlling tumor development in a non-mammary cell-autonomous manner via an immune cell-mediated mechanism. Higher Fbxo10 expression in T cells is associated with Mcs5a increased susceptibility alleles. A common DNA-protein complex bound human and rat sequences containing MCS5A1/Mcs5a1 rs7042509/ss262858675 in electrophoretic mobility shift assays (EMSAs). Lens epithelium-derived growth factor (LEDGF), a stress-response protein, was identified as a candidate to bind both human and rat sequences using DNA-pulldown and mass spectrometry. LEDGF binding was confirmed by LEDGF-antibody EMSA and chromatin immunoprecipitation (ChIP). Ectopic expression of LEDGF/p75 increased luciferase activities of co-transfected reporters containing both human and rat orthologs. Over-expressed LEDGF/p75 increased endogenous FBXO10 mRNA levels in Jurkat cells, a human T-cell line, implying LEDGF may be involved in increasing FBXO10 transcript levels. Oxidative and thermal stress of Jurkat cells increased FBXO10 and LEDGF expression, further supporting a hypothesis that LEDGF binds to a regulatory region of FBXO10 and increases expression during conditions favoring carcinogenesis. We conclude that FBXO10, a candidate breast cancer susceptibility associated gene, is induced by cellular stress and LEDGF may play a role in expression of this gene.

Rat Mcs1b is concordant to the genome-wide association-identified breast cancer risk locus at human 5q11.2 and MIER3 is a candidate cancer susceptibility gene.

Low-penetrance alleles associated with breast cancer risk have been identified in population-based studies. Most risk loci contain either no or multiple potential candidate genes. Rat mammary carcinoma susceptibility 1b (Mcs1b) is a quantitative trait locus on RN02 that confers decreased susceptibility when Copenhagen (COP)-resistant alleles are introgressed into a Wistar Furth (WF)-susceptible genome. Five WF.COP congenic lines containing COP RN02 segments were compared. One line developed an average of 3.4 ± 2.0 and 5.5 ± 3.6 mammary carcinomas per rat ± SD when females were Mcs1b-resistant homozygous and Mcs1b heterozygous, respectively. These phenotypes were significantly different from susceptible genotype littermates (7.8 ± 3.1 mean mammary carcinomas per rat ± SD, P = 0.0001 and P = 0.0413, respectively). All other congenic lines tested were susceptible. Thus, Mcs1b was narrowed to 1.8 Mb of RN02 between genetic markers ENSRNOSNP2740854 and g2UL2-27. Mammary gland-graft carcinoma susceptibility assays were used to determine that donor (P = 0.0019), but not recipient Mcs1b genotype (P = 0.9381), was associated with ectopic mammary carcinoma outcome. Rat Mcs1b contains sequence orthologous to human 5q11.2, a breast cancer susceptibility locus identified in multiple genome-wide association studies. Human/rat MAP3K1/Map3k1 and mesoderm induction early response (MIER; MIER3)/MIER3 are within these orthologous segments. We identified MIER3 as a candidate Mcs1b gene based on 4.5-fold higher mammary gland levels of MIER3 transcripts in susceptible compared with Mcs1b-resistant females. These data suggest that the human 5q11.2 breast cancer risk allele marked by rs889312 is mammary gland autonomous, and MIER3 is a candidate breast cancer susceptibility gene.

Genetically determined inflammatory-response related cytokine and chemokine transcript profiles between mammary carcinoma resistant and susceptible rat strains.

Multiple human breast and rat mammary carcinoma susceptibility (Mcs) alleles have been identified. Wistar Kyoto (WKY) rats are resistant to developing mammary carcinomas, while Wistar Furth (WF) females are susceptible. Gene transcripts at Mcs5a1, Mcs5a2, and Mcs5c are differentially expressed between resistant WKY and susceptible WF alleles in immune-system tissues. We hypothesized that immune-related gene transcript profiles are genetically determined in mammary carcinoma resistant and susceptible mammary glands. Low-density QPCR arrays were used to compare inflammation related genes between mammary carcinoma resistant WKY and susceptible WF females. Mammary gland gene transcript levels predicted to be different based on arrays were tested in independent samples. In total, 20 females per strain were exposed to 7,12-dimethylbenz(a)anthracene (DMBA) to induce mammary carcinogenesis. Twelve age-matched controls per strain without DMBA were included to determine main effects of DMBA-exposure. Significant (ANOVA P ≤ 0.01) effects of strain on mammary gland transcript level were observed for Cx3cl1, Il11ra, Il4, C3, Ccl20, Ccl11, Itgb2, Cxcl12, and Cxcr7. Significant effects of DMBA-exposure were observed for Cx3cl1, Il11ra, Cxcr4, Il4ra, and Il4. Strain and DMBA-exposure interaction effects were significant for Cx3cl1. Transcript levels of Cxcr7 relative to Cxcr4 were modified differently by DMBA in mammary carcinoma resistant and susceptible strains. In conclusion, several genetically-determined differences in cytokine, chemokine, and receptor gene transcript levels were identified between mammary carcinoma susceptible and resistant mammary glands, which may be indicative of cell populations and activities that suppress mammary carcinogenesis in resistant genotypes.

An insulator loop resides between the synthetically interacting elements of the human/rat conserved breast cancer susceptibility locus MCS5A/Mcs5a.

Many low-penetrance breast cancer susceptibility loci are found to be located in non-protein-coding regions, suggesting their involvement in gene expression regulation. We identified the human/rat-conserved breast cancer susceptibility locus MCS5A/Mcs5a. This locus has been shown to act in a non-mammary cell-autonomous fashion through the immune system. The resistant Mcs5a allele from the Wistar-Kyoto (WKy) rat strain consists of two non-protein-coding genetic elements that must be located on the same chromosome to elicit the phenotype. In this study, we show the presence of a conserved higher order chromatin structure in MCS5A/Mcs5a located in between the synthetically interacting genetic elements. The looped elements are shown to be bound by CTCF and cohesin. We identify the downregulation of Fbxo10 expression in T cells as a strong candidate mechanism through which the interacting genetic elements of the resistant Mcs5a allele modulate mammary carcinoma susceptibility. Finally, we show that the human MCS5A polymorphisms associated with breast cancer risk are located at both sides of the looped structure and functionally interact to downregulate transcriptional activity, similar to rat Mcs5a. We propose a mechanistic model for MCS5a/Mcs5a in which a CTCF-mediated insulator loop encompassing the TOMM5/Tomm5 gene, resides in between and brings into closer physical proximity the synthetically and functionally interacting resistant genetic variants.

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.

Physical confirmation and mapping of overlapping rat mammary carcinoma susceptibility QTLs, Mcs2 and Mcs6.

Only a portion of the estimated heritability of breast cancer susceptibility has been explained by individual loci. Comparative genetic approaches that first use an experimental organism to map susceptibility QTLs are unbiased methods to identify human orthologs to target in human population-based genetic association studies. Here, overlapping rat mammary carcinoma susceptibility (Mcs) predicted QTLs, Mcs6 and Mcs2, were physically confirmed and mapped to identify the human orthologous region. To physically confirm Mcs6 and Mcs2, congenic lines were established using the Wistar-Furth (WF) rat strain, which is susceptible to developing mammary carcinomas, as the recipient (genetic background) and either Wistar-Kyoto (WKy, Mcs6) or Copenhagen (COP, Mcs2), which are resistant, as donor strains. By comparing Mcs phenotypes of WF.WKy congenic lines with distinct segments of WKy chromosome 7 we physically confirmed and mapped Mcs6 to ~33 Mb between markers D7Rat171 and gUwm64-3. The predicted Mcs2 QTL was also physically confirmed using segments of COP chromosome 7 introgressed into a susceptible WF background. The Mcs6 and Mcs2 overlapping genomic regions contain multiple annotated genes, but none have a clear or well established link to breast cancer susceptibility. Igf1 and Socs2 are two of multiple potential candidate genes in Mcs6. The human genomic region orthologous to rat Mcs6 is on chromosome 12 from base positions 71,270,266 to 105,502,699. This region has not shown a genome-wide significant association to breast cancer risk in pun studies of breast cancer susceptibility.