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.     

Genetic loci controlling breast cancer susceptibility in the Wistar-Kyoto rat

In this study, the Wistar-Kyoto (WKy) rat was genetically characterized for loci that modify susceptibility to mammary carcinogenesis. We used a genetic backcross between resistant WKy and susceptible Wistar-Furth (WF) rats as a panel for linkage mapping to genetically identify mammary carcinoma susceptibility (Mcs) loci underlying the resistance of the WKy rat. Rats were phenotyped for DMBA-induced mammary carcinomas and genotyped using microsatellite markers. To detect quantitative trait loci (QTL), we analyzed the genome scan data under both parametric and nonparametric distributional assumptions and used permutation tests to calculate significance thresholds. A generalized linear model analysis was also performed to test for interactions between significant QTL. This methodology was extended to identify interactions between the significant QTL and other genome locations. Chromosomes 5, 7, 10, and 14 were found to contain significant QTL, termed Mcs5, Mcs6, Mcs7, and Mcs8, respectively. The WKy alleles of Mcs5, -6, and -8 are associated with mammary carcinoma resistance; the WKy allele of Mcs7 is associated with an increased incidence of mammary cancer. In addition, we identified an interaction between Mcs8 and a region on chromosome 6 termed Mcsm1 (modifier of Mcs), which had no significant main effect on mammary cancer susceptibility in this genetic analysis.     

Analysis of candidate genes included in the mammary cancer susceptibility 1 (Mcs1) region

The rat strain COP is resistant to spontaneous and carcinogen-induced mammary cancer, whereas the strain WF is susceptible. Using genetic linkage analysis of (WF × COP) F₁× WF backcrosses, LC Hsu, LA Shepel and co-workers showed that a region at the centromeric end of Chromosome (Chr) 2 (2q1) segregates with the sensitivity to mammary cancer development. The responsible locus was named Mcs1 (for mammary cancer susceptibility 1). We have developed the chromosome map of the 2q1 region by localizing 18 genes, 4 ESTs, and several anonymous markers, using radiation hybrids and fluorescence in situ hybridization. The region containing Mcs1 was delineated to 2q12–q14. Five of the genes (Mef2c, Map1b, Ccnh, Rasa, Rasgrf2) were assigned to this region and were shown to be expressed in the rat mammary glands, while three possible functional candidate genes, Pi3kr1, Rad17, and Naip, were excluded from the critical region. Since cyclin H, encoded by Ccnh, plays an important role in the control of the cell cycle and since the proteins encoded by Rasa and Rasgrf2 control the activity of the RAS oncoprotein, the corresponding genes appeared as both functional and positional Mcs1 candidates. RT-PCR experiments on RNA extracted from mammary glands of the two rat strains (COP, WF) was done. No amino acid sequence difference was found between the two strains. These results argue against the hypothesis that any of these three genes is Mcs1. Received: 25 September 2000 / Accepted: 15 November 2000     

Mammary cancer susceptibility: human genes and rodent models

Breast cancer is a complex disease, showing a strong genetic component. Several human susceptibility genes have been identified, especially in the last few months. Most of these genes are low-penetrance genes and it is clear that numerous other susceptibility genes remain to be identified. The function of several susceptibility genes indicates that one critical biological pathway is the DNA damage response. However, other pathways certainly play a significant role in breast cancer susceptibility. Rodent models of breast cancer are useful models in two respects. They can help identify new mammary susceptibility genes by taking advantage of the very divergent susceptibilities exhibited by different mouse or rat strains and carrying out relevant genetic analyses. They also provide investigators with experimental systems that can help decipher the mechanism(s) of resistance to mammary cancer. Recent genetic and biological results obtained with mouse and especially with rat strains indicate that (1) numerous quantitative trait loci control mammary cancer susceptibility or resistance, with distinct loci acting in different strains, and (2) distinct resistance mechanisms operate in different rat resistant strains, precocious mammary differentiation being one of these mechanisms.     

Anecdotal,Historical and Critical Commentaries on Genetics: The Utility of Comparative Genetics to Inform Breast Cancer Prevention Strategies

My research seeks to aid in developing approaches to prevent breast cancer. This research evolved from our early empirical studies for discovering natural compounds with anticancer activities, coupled with clinical evaluation to a genetics-driven approach to prevention. This centers on the use of comparative genomics to discover risk-modifying alleles that could help define population and individual risk and also serve as potential prevention drugable targets to mitigate risk. Here, we initially fine map mammary cancer loci in a rat carcinogenesis model and then evaluate their human homologs in breast cancer case-control association studies. This approach has yielded promising results, including the finding that the compound rat QTL Mcs5a''s human homologous region was associated with breast cancer risk. These and related findings have the potential to yield advancements both in translation-prevention research and in basic molecular genetics.WRITING this Perspectives for Genetics allows me to examine how a cancer biologist focused on cancer prevention morphed into a practicing geneticist. In addition, it allows me to review a decade of our investigations into the complexity of the genetic risk to breast cancer development using comparative genomics.Our comparative genomic strategy consists of genetically identifying mammary cancer risk loci using fine mapping studies in a rat mammary carcinogenesis model. Human homologs of these loci are then evaluated in human breast cancer association studies for their potential to modify risk. This genetics approach provides an integrated discovery platform to identify and mechanistically characterize novel breast cancer risk alleles. We predict that this platform will serve as a foundation for a cancer prevention drug development pipeline.My early work focused on the etiology and prevention of breast cancer. It is work on these interrelated areas that led me to investigate breast cancer genetics. While studying the etiology of breast cancer after joining the faculty of the University of Wisconsin, my interest targeted early events in the etiology of cancer. These range from altering the metabolic activation of environmental xenobiotics to metabolites capable of adducting DNA to destroying clones premalignant cells. At the time we began work in this area, cancer chemoprevention was an emerging field that was assumed to be less complex than cancer therapy. This was, in part, based on the fact that normal and premalignant tissues were genetically more stable than cancer cells and thus less likely to develop resistance to anticancer drugs.Our chemoprevention studies focused on a novel class of nontoxic monoterpenes widely found in the essential oils of fruits. These compounds were found to have both preventive and therapeutic anticancer activities in being able to inhibit both premalignant and malignant cells. Our lead compound was limonene, found in orange peel oil, and the first monoterpene we entered into FDA-approved clinical trials was perillyl alcohol (POH), found originally in lavender oil. For expediency, our first trial was a therapeutic one. This therapeutic phase I trial showed limited promising results (Ripple et al. 2000). We later discovered that POH inhibited the antiapoptotic ability of cancers via a calcium channel interaction that led to the downregulation of NFκB (Berchtold et al. 2005). This mechanism of action could underlie the cytostatic and cytotoxic actions of POH toward both premalignant and malignant cells.The monoterpenes and POH were found through empirical screening. Like the monoterpenes, many chemopreventive and therapeutic agents are found to be of low overall efficacy. Many also have undesirable toxicity, in part due to the lack of target specificity. As such, we felt the need to develop nonempirical methods to develop prevention strategies and drugs.To develop chemopreventative agents for common diseases, we sought an approach that would identify both appropriate drug targets and high-risk populations. For example, we aimed to develop prevention strategies for the large number of individuals at risk for breast cancer but not those who specifically carried the rare but highly penetrant susceptibility alleles of the breast cancer genes such as Brca1 and -2; these and other highly penetrant breast cancer risk alleles collectively account for <25% of inherited breast cancer risk in humans (Pharoah et al. 2008).We thus sought to identify moderately penetrant breast cancer susceptibility alleles that were common (high population frequency). Ten years ago it was difficult to identify such loci directly in human populations. In fact, most association studies at that time were based on a “candidate gene” approach; these studies were rarely successful (Pharoah et al. 2007). We thus adapted a comparative genomics strategy in which such loci are identified in a model organism using a nonbiased linkage approach and then evaluated in humans. We chose what we believe is the in vivo breast cancer model most closely related to the human—the rat.The rat, in contrast to the mouse and like the human, develops a spectrum of hormonally responsive and nonresponsive breast cancers. Importantly, almost all rat and human cancers have a ductal cell origin (Gould 1995). At the time we began this research, however, the rat had far fewer genetic resources and tools than the mouse (Gould 1995). This can be illustrated by our need to use a M13 minisatellite marker to identify our first rat mammary susceptibility QTL (Hsu et al. 1994). Over the course of this research and subsequent studies, rat geneticists have substantially narrowed this technology gap (see Aitman et al. 2008). For example, in pursuing this project we developed a technology that produced the first gene inactivation (“knockout”) rat models (Zan et al. 2003).

Comparative genetics studies:

The first major results of these genomewide comparative studies were published by Shepel et al. (1998) in Genetics. In this study we crossed two rat strains with large differences in their susceptibility to the induction of mammary carcinomas by the chemical carcinogen dimethylbenzanthracene (DMBA). The susceptible strain was the Wistar-Furth (WF) rat, while the resistance strain was the Copenhagen (COP) rat. F₁ hybrid rats were backcrossed (WF × COP) F₁ × WF or intercrossed (F₁ × F₁). Large groups of these rats were orally gavaged with DMBA, and the average number of mammary carcinomas per rat was quantified at necropsy. Rats were also genotyped using microsatellite markers, which had become available for the rat in the 1990s.The QTL genetically identified in this study accounted for most of the genetic variance controlling susceptibility to mammary cancer by identifying the Mammary carcinoma susceptibility (Mcs) loci—Mcs1, -2, -3, and -4. The COP allele of Mcs1, -2, and -3 conferred resistance while Mcs4 conferred an increased susceptibility to mammary cancer development. This study demonstrated the ability to use the rat model to identify the major COP vs. WF polymorphic loci controlling susceptibility. These loci interacted in an additive manner. Interestingly, the almost completely mammary cancer-resistant COP rat strain was shown to carry a polymorphic allele at the Mcs4 locus predicted to increase mammary cancer risk.In extending this study, we asked whether other mammary cancer-resistant strains varied at polymorphic mammary cancer susceptibility loci shared with those genetically identified in the WF × COP cross. A similar analysis was performed by conducting a QTL analysis of a cross between WF and a second resistant strain Wistar-Kyoto (WKy). In this backcross analysis we genetically identified four loci that accounted for most of the genetic variance associated with the susceptibility phenotype. As with the COP cross, the WKy cross identified three loci in which the WKy allele contributed to resistance and one locus at which the WKy allele contributed to increased susceptibility (Lan et al. 2001). Of these four WKy loci, only one broadly overlapped with those identified in the COP × WF cross, i.e., Mcs2 (COP) with Mcs6 (WKy). This study also used a novel statistical approach developed by our statistical collaborator, Christina Kendziorski, to identify alleles with no main effect that modify QTL with main effects. Mcs-modifier 1 (Mcsm1) was the most strongly supported locus of this class. The WKy allele of this locus fully negated the effects of the resistance conferred by the WKy allele of the Mcs8 QTL. Thus it appeared that there could be a large number of polymorphic loci in rats that could contribute to mammary cancer risk.It is important to keep in mind that genetically identified QTL are the product of statistical modeling and analysis of segregating populations from crosses. It is thus critical that their existence be confirmed in more homogeneous genetic material. An established method for QTL validation is to breed and phenotype congenic animals carrying only the region surrounding the QTL allele of interest on an alternative genetic background. So far we have generated and characterized six of the eight candidate WKy and COP QTL by genetically introgressing them onto the WF background. All six have the phenotype predicted by our quantitative models.Most congenic substitutions include tens of megabases encompassing the introgressed allele. The next step is to fine map this congenic interval to first determine whether this interval harbors more than one independent susceptibility locus. In addition, the fine mapping process allows for an increased genomic resolution of the locus and thus a more limited set of candidates. We have fine mapped two Mcs loci–Mcs1 (COP) and Mcs5 (WKy). Each was found to be complex, containing at least three separable subloci termed Mcs1a, -b, -c and Mcs5a, -b, -c. In the case of Mcs1, all three identified loci within it contributed to the cancer resistance phenotype of Mcs1. This led us to speculate that this apparent clustering might be biologically “random”; their strong-combined phenotype allowed us to readily identify Mcs1 over the experimental background. In contrast, the Mcs5 also had at least three subloci, Mcs5a, -b, and -c, but two of these, a and c, contribute to resistance while b confers an increased sensitivity. Each of the three had similar absolute relative risk (RR) contributions. If they interact in a purely additive manner, it might have been difficult to identify Mcs5. However, Mcs5 had the strongest of LOD scores of any identified locus in the WKy cross (Lan et al. 2001). When we explored the interaction of the alleles at the Mcs5 loci, we found complex epistatic interactions. The strongest was the complete neutralization of the effect of the sensitive WKy allele of Mcs5b by the resistant WKy allele of Mcs5a (Samuelson et al. 2005).It is interesting to explore an alternative hypothesis that suggests that the clustering of mammary cancer susceptibility alleles arise from evolutionary selection. Data supporting such a possibility in rodents has been published by Petkov et al. (2005). Their findings suggest that alleles controlling certain phenotypes cluster to assure joint inheritance, in that in concert with one another, they provide for an enhanced survival advantage. This could account for the clustering of risk-related genes at the Mcs1 and Mcs5 QTL.Many of the most comprehensive published mammalian fine-mapping studies achieve mapping resolutions in the order of several megabases. Such intervals, while carrying a limited number of genes, often require choosing one or more candidate genes for intensive study. These are usually chosen on the basis of how they might functionally relate to the specific disease risk under investigation. This negates the potential of positional cloning to identify an unbiased candidate. As mentioned above, experience suggests that functional candidate selection rarely identifies disease-specific modifier genes. For example, in breast cancer, when 120 such published candidates (710 SNPs) were rigorously evaluated, none met minimal statistical significance in a study of a large population of women in a breast cancer case-control study (Pharoah et al. 2007).We explored the ability of ultrafine mapping to annotate the Mcs5a locus. We mapped this locus to >100-kb resolution by phenotyping congenic rats recombinant within this locus. We found it to contain two elements. The WKy allele of each element by itself failed to elicit a mammary cancer phenotype; however, when combined, the resistance phenotype was obvious. These elements, termed Mcs5a1 and Mcs5a2, synthetically interact, making Mcs5a one of the first-identified compound QTL in mammals. Because Mcs5a acts in a semidominant manner, we could use heterozygous congenic recombinants to ask whether both elements of Mcs5a needed to lie in cis on the same chromosome, or could they interact in trans from separate homologs. They interact only in cis (Samuelson et al. 2007). Another interesting observation arising from the fine mapping of Mcs5a is that it localizes to noncoding DNA. All four Mcs loci that we have fine mapped to high resolution are localized to noncoding DNA (in progress).The observations that the rat compound locus Mcs5a consists of two synthetically interacting elements separated by ∼50-60 kb (based on the human sequence), interact only when on the same chromosome, and are noncoding suggest the hypothesis that they may be localized in closer proximity than suggested by the linear genomic distance that separates them. Recent observations in our laboratory using chromosome confirmation capture suggest that most of the sequences between these elements form a CTCF-mediated loop bringing both elements in close physical proximity to each other. The ability of this compound locus to control local and interchromosomal gene expression is being studied (in progress).To determine whether our findings in our rat model could be extended to women, we next asked whether the human ortholog of Mcs5a (-a1 and -a2) could influence breast cancer risk. In contrast to the method of searching for modifier genes using genomewide association studies (GWAS), we restricted our search to an ∼100-kb region of the human genome. Focusing on this orthologous locus defined by comparative genomics vastly reduced the number of SNP-tagged alleles needed for testing for association, greatly reducing the statistical penalty for multiple testing. We tested several SNPs in the orthologous MCS5A1 and -5A2 regions of the human genome in a total of ∼12,000 women in a breast cancer case-control study. We found that a tagged SNP in both MCS5A1 and -5A2 was significantly associated with risk to breast cancer in this population of women. The minor allele of SNP rs56476643 (MCS5A1) acts in a recessive manner to increase risk. Its allele frequency is 25% and it increases risk in homozygous women by 19%. In contrast, the minor allele of MCS5A2 (rs2182317) has an allele frequency of 13% and acts in a dominant manner to reduce by 14% the risk of breast cancer in the 24% of women carrying one or two copies of this allele (Samuelson et al. 2007).Not only does this human study support the use of comparative genomics to identify human cancer risk modifier alleles, it also extends the resolution obtained in the rat in localizing the two genetic elements of the Mcs5a allele. The rat localizes Mcs5a1 and -a2 to 32 and 84 kb, respectively, while the human studies resolved these determinants to 5.7 kb and 16.8 kb (Samuelson et al. 2007). Thus, we have demonstrated a clear advantage in using comparative genomics to localize target regions within QTL.Both MCS5A1 and -5A2 have similar allele frequencies and genetic penetrance (relative risk) as do most breast cancer alleles identified by GWAS studies. However, unlike alleles identified by GWAS studies, those identified by comparative genomics also provide in vivo models to functionally characterize risk alleles. For example, it is often assumed that breast cancer modifiers are likely to act within breast tissue to modulate risk. Using the rat as a model we have been able to show that Mcs5a, a noncoding allele, acts to differentially regulate its neighboring FBXO10 gene in immune but not mammary tissues (Samuelson et al. 2007).It is also intriguing to consider the observation that breast cancer risk-associated alleles such as Mcs5a1 and -5a2 are either conserved over millions of evolutionary years or are highly mutable and functionally neutral, suggesting that these alleles do not significantly reduce fitness. If so, one then speculates that they would make good targets for chemoprevention drugs by possessing low toxicity and as such a good therapeutic index. In particular, converting sensitive to resistant allelic function with drug therapy would mimic the conserved resistance allele that persists in the human population and should therefore show a low side-effects profile.Our current research on these genetically identified Mcs loci focuses on molecular, cellular, and organismal mechanisms by which they modify risk. Not only will these investigations provide insight into the function of each noncoding Mcs locus, but collectively they will provide a mechanistic framework to facilitate integrative genetic studies of the plethora of polymorphic risk loci identified by GWAS in multiple diseases.     

Genetic determination of susceptibility to estrogen-induced mammary cancer in the ACI rat: mapping of Emca1 and Emca2 to chromosomes 5 and 18

Hormonal, genetic, and environmental factors play major roles in the complex etiology of breast cancer. When treated continuously with 17beta-estradiol (E2), the ACI rat exhibits a genetically conferred propensity to develop mammary cancer. The susceptibility of the ACI rat to E2-induced mammary cancer appears to segregate as an incompletely dominant trait in crosses to the resistant Copenhagen (COP) strain. In both (ACI x COP)F(2) and (COP x ACI)F(2) populations, we find strong evidence for a major genetic determinant of susceptibility to E2-induced mammary cancer on distal rat chromosome 5. Our data are most consistent with a model in which the ACI allele of this locus, termed Emca1 (estrogen-induced mammary cancer 1), acts in an incompletely dominant manner to increase both tumor incidence and tumor multiplicity as well as to reduce tumor latency in these populations. We also find evidence suggestive of a second locus, Emca2, on chromosome 18 in the (ACI x COP)F(2) population. The ACI allele of Emca2 acts in a dominant manner to increase incidence and decrease latency. Together, Emca1 and Emca2 act independently to modify susceptibility to E2-induced mammary cancer.     

Genetic identification of distinct loci controlling mammary tumor multiplicity, latency, and aggressiveness in the rat

The rat is considered an excellent model for studying human breast cancer. Therefore, understanding the genetic basis of susceptibility to mammary cancer in this species is of great interest. Previous studies based on crosses involving the susceptible strain WF (crossed with the resistant strains COP or WKY) and focusing on tumor multiplicity as the susceptibility phenotype led to the identification of several loci that control chemically induced mammary cancer. The present study was aimed to determine whether other loci can be identified by analyzing crosses derived from another susceptible strain on the one hand, and by including phenotypes other than tumor multiplicity on the other hand. A backcross was generated between the susceptible SPRD-Cu3 strain and the resistant WKY strain. Female progeny were genotyped with microsatellite markers covering all rat autosomes, treated with a single dose of DMBA, and phenotyped with respect to tumor latency, tumor multiplicity, and tumor aggressiveness. Seven loci controlling mammary tumor development were detected. Different loci control tumor multiplicity, latency, and aggressiveness. While some of these loci colocalize with loci identified in crosses involving the susceptible strain WF, new loci have been uncovered, indicating that the use of distinct susceptible and resistant strain pairs will help in establishing a comprehensive inventory of mammary cancer susceptibility loci.     

Multiple Susceptibility Loci for Radiation-Induced Mammary Tumorigenesis in F2[Dahl S x R]-Intercross Rats

Although two major breast cancer susceptibility genes, BRCA1 and BRCA2, have been identified accounting for 20% of breast cancer genetic risk, identification of other susceptibility genes accounting for 80% risk remains a challenge due to the complex, multi-factorial nature of breast cancer. Complexity derives from multiple genetic determinants, permutations of gene-environment interactions, along with presumptive low-penetrance of breast cancer predisposing genes, and genetic heterogeneity of human populations. As with other complex diseases, dissection of genetic determinants in animal models provides key insight since genetic heterogeneity and environmental factors can be experimentally controlled, thus facilitating the detection of quantitative trait loci (QTL). We therefore, performed the first genome-wide scan for loci contributing to radiation-induced mammary tumorigenesis in female F2-(Dahl S x R)-intercross rats. Tumorigenesis was measured as tumor burden index (TBI) after induction of rat mammary tumors at forty days of age via ¹²⁷Cs-radiation. We observed a spectrum of tumor latency, size-progression, and pathology from poorly differentiated ductal adenocarcinoma to fibroadenoma, indicating major effects of gene-environment interactions. We identified two mammary tumorigenesis susceptibility quantitative trait loci (Mts-QTLs) with significant linkage: Mts-1 on chromosome-9 (LOD-2.98) and Mts-2 on chromosome-1 (LOD-2.61), as well as two Mts-QTLs with suggestive linkage: Mts-3 on chromosome-5 (LOD-1.93) and Mts-4 on chromosome-18 (LOD-1.54). Interestingly, Chr9-Mts-1, Chr5-Mts-3 and Chr18-Mts-4 QTLs are unique to irradiation-induced mammary tumorigenesis, while Chr1-Mts-2 QTL overlaps with a mammary cancer susceptibility QTL (Mcs 3) reported for 7,12-dimethylbenz-[α]antracene (DMBA)-induced mammary tumorigenesis in F2[COP x Wistar-Furth]-intercross rats. Altogether, our results suggest at least three distinct susceptibility QTLs for irradiation-induced mammary tumorigenesis not detected in genetic studies of chemically-induced and hormone-induced mammary tumorigenesis. While more study is needed to identify the specific Mts-gene variants, elucidation of specific variant(s) could establish causal gene pathways involved in mammary tumorigenesis in humans, and hence novel pathways for therapy.     

Rat Chromosome 2: assignment of the genes encoding cyclin B1, interleukin 6 signal transducer, and proprotein convertase 1 to the Mcs1-containing region and identification of new microsatellite markers

The rat Chromosome (Chr) 2 harbors several genes controlling tumor growth or development, blood pressure, and non-insulin-dependent diabetes mellitus. We report that the region (2q1) containing the mammary susceptibility cancer gene Mcs1 also harbors the genes encoding cyclin B1, interleukin 6 signal transducer (gp130), and proprotein convertase 1. We also generated 13 new anonymous microsatellite markers from Chr 2-sorted DNA. These markers, as well as a microsatellite marker in the cyclin B1 gene, were genetically mapped in combination with known markers. A cyclin B1-related gene was also cytogenetically assigned to rat Chr 11q22-q23. Received: 21 July 1998 / Accepted: 28 August 1998     

Breast cancer gene discovery

Many important advances have been made in the past decade in understanding breast cancer at the molecular level, and two important high-penetrance breast cancer genes--BRCA1 and BRCA2--have been identified. However, germline mutations in these two genes are responsible for only a minority (approximately 5%) of all breast carcinomas, and the genes responsible for the majority of breast cancer cases remain to be identified. There is evidence that there are additional high-to-moderate-penetrance breast cancer susceptibility genes but, given the high degree of molecular heterogeneity in breast carcinomas, it is likely that each of these genes is responsible for only a subset of cases. There are also many candidate low-penetrance breast cancer genes and many more are likely to be identified. In addition to germline, and somatic, sequence alterations, epigenetic changes in many genes are likely to play an important role in the pathobiology of breast cancer. Recently developed genomic technologies and the completion of the human genome sequence provide us with powerful tools to identify novel candidate breast cancer genes that could play an important role in breast tumourigenesis.     

Hepatocellular carcinoma as a complex polygenic disease. Interpretive analysis of recent developments on genetic predisposition

The different frequency of hepatocellular carcinoma (HCC) in humans at risk suggests a polygenic predisposition. However, detection of genetic variants is difficult in genetically heterogeneous human population. Studies on mouse and rat models identified 7 hepatocarcinogenesis susceptibility (Hcs) and 2 resistance (Hcr) loci in mice, and 7 Hcs and 9 Hcr loci in rats, controlling multiplicity and size of neoplastic liver lesions. Six liver neoplastic nodule remodeling (Lnnr) loci control number and volume of re-differentiating lesions in rat. A Hcs locus, with high phenotypic effects, and various epistatic gene-gene interactions were identified in rats, suggesting a genetic model of predisposition to hepatocarcinogenesis with different subset of low-penetrance genes, at play in different subsets of population, and a major locus. This model is in keeping with human HCC epidemiology. Several putative modifier genes in rodents, deregulated in HCC, are located in chromosomal segments syntenic to sites of chromosomal aberrations in humans, suggesting possible location of predisposing loci. Resistance to HCC is associated with lower genomic instability and downregulation of cell cycle key genes in preneoplastic and neoplastic lesions. p16(INK4A) upregulation occurs in susceptible and resistant rat lesions. p16(INK4A)-induced growth restraint was circumvented by Hsp90/Cdc37 chaperons and E2f4 nuclear export by Crm1 in susceptible, but not in resistant rats and human HCCs with better prognosis. Thus, protective mechanisms seem to be modulated by HCC modifiers, and differences in their efficiency influence the susceptibility to hepatocarcinogenesis and probably the prognosis of human HCC.     

Susceptibility to neoplastic and non-neoplastic pulmonary diseases in mice: genetic similarities

Chronic inflammation predisposes toward many types of cancer. Chronic bronchitis and asthma, for example, heighten the risk of lung cancer. Exactly which inflammatory mediators (e.g., oxidant species and growth factors) and lung wound repair processes (e.g., proangiogenic factors) enhance pulmonary neoplastic development is not clear. One approach to uncover the most relevant biochemical and physiological pathways is to identify genes underlying susceptibilities to inflammation and to cancer development at the same anatomic site. Mice develop lung adenocarcinomas similar in histology, molecular characteristics, and histogenesis to this most common human lung cancer subtype. Over two dozen loci, called Pas or pulmonary adenoma susceptibility, Par or pulmonary adenoma resistance, and Sluc or susceptibility to lung cancer genes, regulate differential lung tumor susceptibility among inbred mouse strains as assigned by QTL (quantitative trait locus) mapping. Chromosomal sites that determine responsiveness to proinflammatory pneumotoxicants such as ozone (O3), particulates, and hyperoxia have also been mapped in mice. For example, susceptibility QTLs have been identified on chromosomes 17 and 11 for O3-induced inflammation (Inf1, Inf2), O3-induced acute lung injury (Aliq3, Aliq1), and sulfate-associated particulates. Sites within the human and mouse genomes for asthma and COPD phenotypes have also been delineated. It is of great interest that several susceptibility loci for mouse lung neoplasia also contain susceptibility genes for toxicant-induced lung injury and inflammation and are homologous to several human asthma loci. These QTLs are described herein, candidate genes are suggested within these sites, and experimental evidence that inflammation enhances lung tumor development is provided.     

Most lung and colon cancer susceptibility genes are pair-wise linked in mice, humans and rats

Genetic predisposition controlled by susceptibility quantitative trait loci (QTLs) contributes to a large proportion of common cancers. Studies of genetics of cancer susceptibility, however, did not address systematically the relationship between susceptibility to cancers in different organs. We present five sets of data on genetic architecture of colon and lung cancer susceptibility in mice, humans and rats. They collectively show that the majority of genes for colon and lung cancer susceptibility are linked pair-wise and are likely identical or related. Four CcS/Dem recombinant congenic strains, each differing from strain BALB/cHeA by a different small random subset of ±12.5% of genes received from strain STS/A, suggestively show either extreme susceptibility or extreme resistance for both colon and lung tumors, which is unlikely if the two tumors were controlled by independent susceptibility genes. Indeed, susceptibility to lung cancer (Sluc) loci underlying the extreme susceptibility or resistance of such CcS/Dem strains, mapped in 226 (CcS-10 x CcS-19)F2 mice, co-localize with susceptibility to colon cancer (Scc) loci. Analysis of additional Sluc loci that were mapped in OcB/Dem strains and Scc loci in CcS/Dem strains, respectively, shows their widespread pair-wise co-localization (P = 0.0036). Finally, the majority of published human and rat colon cancer susceptibility genes map to chromosomal regions homologous to mouse Sluc loci. 12/12 mouse Scc loci, 9/11 human and 5/7 rat colon cancer susceptibility loci are close to a Sluc locus or its homologous site, forming 21 clusters of lung and colon cancer susceptibility genes from one, two or three species. Our data shows that cancer susceptibility QTLs can have much broader biological effects than presently appreciated. It also demonstrates the power of mouse genetics to predict human susceptibility genes. Comparison of molecular mechanisms of susceptibility genes that are organ-specific and those with trans-organ effects can provide a new dimension in understanding individual cancer susceptibility.     

Molecular pathology of breast apocrine carcinomas: a protein expression signature specific for benign apocrine metaplasia

Breast cancer is a heterogeneous disease that encompasses a wide range of histopathological types including: invasive ductal carcinoma, lobular carcinoma, medullary carcinoma, mucinous carcinoma, tubular carcinoma, and apocrine carcinoma among others. Pure apocrine carcinomas represent about 0.5% of all invasive breast cancers according to the Danish Breast Cancer Cooperative Group Registry, and despite the fact that they are morphologically distinct from other breast lesions, there are at present no standard molecular criteria available for their diagnosis. In addition, the relationship between benign apocrine changes and breast carcinoma is unclear and has been a matter of discussion for many years. Recent proteome expression profiling studies of breast apocrine macrocysts, normal breast tissue, and breast tumours have identified specific apocrine biomarkers [15-hydroxyprostaglandin dehydrogenase (15-PGDH) and hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase)] present in early and advanced apocrine lesions. These biomarkers in combination with proteins found to be characteristically upregulated in pure apocrine carcinomas (psoriasin, S100A9, and p53) provide a protein expression signature distinctive for benign apocrine metaplasias and apocrine cystic lesions. These studies have also presented compelling evidence for a direct link, through the expression of the prostaglandin degrading enzyme 15-PGDH, between early apocrine lesions and pure apocrine carcinomas. Moreover, specific antibodies against the components of the expression signature have identified precursor lesions in the linear histological progression to apocrine carcinoma. Finally, the identification of proteins that characterize the early stages of mammary apocrine differentiation such as 15-PGDH, HMG-CoA reductase, and cyclooxygenase 2 (COX-2) has opened a window of opportunity for pharmacological intervention, not only in a therapeutic manner but also in a chemopreventive setting. Here we review published and recent results in the context of the current state of research on breast apocrine cancer.