Etiology
In developing its priorities for research funding, the Breast Cancer Research Council identified study into potential causes of breast cancer (etiology) as a critical topic. A major goal of the CBCRP is to reduce the incidence and morbidity of breast cancer among women in California. Understanding the etiology and pathogenesis of breast cancer was deemed an essential first step toward the development of effective preventive interventions.
It is well known that breast cancer affects some groups in the population at higher rates than others. A better understanding of who gets breast cancer and who doesn't will lead to clues about risks and prevention. Under the broad topic of etiology, the Council envisioned a variety of possible studies, ranging from laboratory research studying potential cancer-causing agents and genetic abnormalities in breast cancer cells or tissue, to large scale population studies that might examine dietary, hormonal, and environmental factors associated with an increased risk of breast cancer. The Council thought that these would be important first steps in enhancing our ability to recommend interventions for larger scale clinical testing. The Council deemed this a high priority area because of its potential to shed light on the causes of breast cancer and the development of preventive intervention strategies in the future. It was felt that federal funding for preventive interventions had been relatively modest, and that this would be an innovative area for the CBCRP to support.
CBCRP received many high quality applications in this area and funded projects in almost all of the areas included in this topic. Two projects are examining genetic aspects of breast cancer (Groffen; Henderson), one exploring two candidate tumor suppressor genes, and the second examining the estrogen receptor gene and estrogen metabolism. Two other studies are looking at potential carcinogens and the risk of breast cancer (Creek; Goth-Goldstein). In these studies, xenoestrogens and heterocyclic aromatic amines (both substances which women are exposed to) are being examined for their association with breast cancer risk using tissue samples and epidemiological techniques. Finally, there are several epidemiological studies examining dietary and lifestyle factors in relation to breast cancer (Carpenter; Enger; Ingles; Wu).
ABSTRACTS
Two Candidate Breast Cancer Genes on Chromosome 17
John Groffen, Ph.D.
Childrens Hospital of Los Angeles
Most, if not all human cancers are caused by mutations in specific genes. These mutations can be inherited, in the case of familial cancers, but more often develop during the lifetime of an individual. The mutations lead to the development of tumors, either directly or indirectly.
Human genetic material is packed into 22 pairs of chromosomes, in addition to the gender specific chromosomes. When the chromosomes of breast cancer tissues are examined, it is frequently observed that either the number of chromosomes, the structure of certain chromosomes, or both are different than those found in normal breast tissues.
It has been demonstrated that some breast cancer tumors are missing a very small segment of one of the two human chromosomes 17 at a location called 17p13.3. It is normal for human cells to contain two copies of each chromosome and normally both copies of every gene are functional. There is evidence that a gene which is important for the suppression of the development of cancer is located within the missing segment on one copy of chromosome 17 in the breast cancer cells, and that the other copy of this gene has been destroyed, mutated or inactivated by other events. This would leave the breast cancer cells with no normal copy of this gene. Such a so-called tumor suppressor gene is directly or indirectly involved in the control of cellular growth and lack of it is likely to be inherently connected to the development of cancer.
Two genes named CRK and ABR were localized by us to this region of chromosome 17 and one copy of each appears to be missing in certain breast cancer cells. In animals, the CRK gene is involved in cancer. ABR is closely related to a gene which is responsible for human leukemia. Other genes which resemble ABR can cause the development of solid tumors.
Genes are responsible for the production of proteins. If a gene is damaged, it may make a wrong protein, or no protein at all. According to our initial experiments, the proteins which should be made by the CRK and ABR genes are either present in low quantities or are absent altogether from certain breast cancer cells. We will extend this work to determine whether the CRK and ABR proteins are missing from a broader range of breast cancer tumors. In addition, we will investigate the reasons why no protein is made in the breast cancer cells by examining the structure of the copy of ABR and/or CRK gene that still remains.
If we discover that one copy of the ABR and/or CRK gene is specifically damaged while the other copy is missing altogether in breast cancer tissues but not in the surrounding normal tissue, the discovery is likely to have a far reaching impact in breast cancer research. It will immediately lead to the development of improved methods of diagnosis and provide the basis for the development of new therapies for breast cancer.
Metabolism of Environmental Chemicals as Breast Cancer Risk
Regine Goth-Goldstein, Ph.D.
Lawerence Berkeley National Laboratory
Analysis of mutations in breast tumors suggests that environmental chemicals such as polycyclic aromatic hydrocarbons (PAHs) might be involved in the cause of breast cancer. PAHs are ubiquitous in the environment, and are ingested and inhaled continuously. PAHs act as carcinogens only after being activated in the body. The balance of PAH-activating and detoxifying enzymes determines the amount of binding of PAHs to DNA, which in turn determines the risk of cancer initiation. The activity of these enzymes is determined by genetic and environmental factors. The synthesis of activating enzymes can be induced by PAHs themselves and other environmental toxins, such as dioxin. The interindividual variations in activity of activating and detoxifying enzymes contribute to individual susceptibility to cancer of the lung, colon and bladder. It is proposed to determine if the amount of PAH-activating and detoxifying enzymes in breast tissue represents a risk factor for breast cancer. If this is the case, women with increased susceptibility could be identified and certain preventive measures could be taken, such as more frequent examination, changes in lifestyle to reduce PAH exposure, and possibly changes in diet to alter the balance of PAH- metabolizing enzymes.
These hypotheses will be tested by examining the two major enzymes involved in PAH metabolism: the activating enzyme, cytochrome P4501A1, encoded by the gene CYPlAl, and the detoxifying enzyme glutathione S-transferase, encoded by the gene GSTM1. Variability in the activity of these enzymes is due in part to genetic variation, and in part to modification of gene expression by environmental agents. The presence or absence of the GSTM1 gene, and expression of CYP1A1 will be determined in breast tissue. A collection of about one hundred microscopically normal breast tissue specimens from mastectomy patients or from reduction mammoplasties will be analyzed. CYP1A1 expression (determined by quantitative reverse transcription polymerase chain reaction) and GSTM1 genotype (determined by PCR) will be compared in individuals with breast cancer and healthy individuals. In addition, the correlation of PAH-DNA adduct formation and PAH-metabolizing enzymes will be examined in vitro by treating cells from specimens with different enzyme activity with a model compound and then measuring DNA abducts. These studies should indicate if there is a trend of higher CYP1A1 expression and/or absence of GSTM1 in breast cancer patients.
Xenoestrogens and Genetic Damage in Breast Cancer
Moire Robertson Creek, Ph.D.
Stanford Research Institute International
Estrogens are a class of hormones critical to female sexual development and functioning, but, paradoxically, strong evidence links them with the induction of breast cancer. Precisely which estrogen is responsible is unknown, but high levels of 16-hydroxy-estrone (16 -OHE), a metabolite of the estrogen estradiol, are strongly correlated with the susceptibility to and presence of breast cancer in human and animals. Additional evidence suggests that certain dietary and environmental factors may alter estrogen production or metabolism and thereby function as “xenoestrogens.” Xenoestrogens are broadly defined as substances that alter normal estrogen homeostasis (regulation) and function. For example, dietary substances found in cruciferous vegetables, e.g., broccoli or cauliflower, lower the effective levels of 16 -OHE and decrease mammary tumor incidence by inducing competing metabolic pathways. On the other hand, ethanol is thought to increase tumor formation by enhancing metabolic pathways that form 16-OHE. Definitive studies showing the relationship between 16-OHE levels, enzyme induction or inhibition, and xenoestrogen exposure have not been conducted and are the subject of this proposal. These studies will define the relative ability of estradiol and its metabolites (e.g., 16-OHE) to alter genetic material and will examine how xenoestrogens influence the formation of these genotoxic compounds by altering the metabolic pathways leading to their formation. This knowledge may ultimately lead to effective methods for identifying susceptible populations, detecting early stages of breast cancer, reducing preventable causes of breast cancer, and designing chemicals that will help prevent development of breast cancer. We will address two priority issues of the Breast Cancer Research Program: the role of genotoxic agents in the origin of breast cancer, and early detection of breast cancer.
We hypothesize that 16-OHE may initiate tumor formation by interacting directly with proteins and DNA; thereby leading to chromosomal breakage or loss. Furthermore, since the metabolism of estradiol to 16-OHE is dependent upon xeno-estrogen-inducible metabolic enzyme systems, we also hypothesize that the formation of this metabolite may be affected by xenoestrogens. Specifically, we will study the chromosome-damaging effects of estradiol and its metabolites, investigate covalent binding of estradiol and its metabolites to large molecules both in cell cultures and in breast tissue of animals with varying susceptibilities to breast cancer, study the effect of selected xenoestrogens on the profile of covalent binding, and examine how xenoestrogens alter estradiol metabolism and estradiol-induced genetic damage.
Physical Activity: Impact on Hormones and Breast Cancer Risk
Catherine L. Carpenter, Ph.D.
University of Southern California
Evidence from epidemiologic studies suggest that women who engage in strenuous physical activity are at a reduced risk of breast cancer. Despite the emergence of this relationship, several population subgroups such as postmenopausal women, and premenarcheal girls have not been well studied. The objectives of the proposed research are: (1) to assess the effect of lifetime physical activity on breast cancer risk in postmenopausal women, and (2) to further clarify how physical activity influences age at puberty and ovarian hormone patterns in the years immediately following the onset of puberty. Results from this study could further clarify the link between lifestyle factors and hormonal events among adolescent girls. Furthermore, identification of physical activity as a possible protective factor among postmenopausal women could provide an intervention tool to assist the effort to reduce breast cancer incidence among women in California.
Possible biological mechanisms have been suggested to explain the influence of physical activity on breast cancer risk. Physical activity is one of several breast cancer risk factors that affect exposure to endogenous hormones. Strenuous physical activity has been shown to increase the age at menarche, menstrual cycle length, and number of anovulatory cycles (a menstrual cycle without release of an ovum). Furthermore, moderate physical activity has been linked to the reduction of frequency of ovulatory menstrual cycles.
Data from a matched case-control study of hormone replacement therapy and breast cancer risk among postmenopausal Caucasian, women age 55 or older will be utilized to address the first objective. A total of 1510 case-control pairs (women who have had breast cancer matched with women who have not had breast cancer matched on age, race/ethnicity, and neighborhood) provided detailed information on their lifetime participation in physical activities. Appropriate statistical techniques will judge the possible effect of physical activity on breast cancer risk.
Data from a cohort study of 1200 predominately Hispanic school children from the 4th through the 7th grades will be utilized to address the second objective. Data on physical activity and menarche status is available for 808 girls. Hormonal status and physical activity data has been collected on 345 girls. Again, appropriate statistical techniques will be used to estimate the effect of physical activity on hormonal status.
Results from this study should further clarify the link between lifestyle factors and hormonal events among adolescent girls. Furthermore, identification of physical activity as a possible protective factor among postmenopausal women would provide a sound basis for an intervention to assist the effort to reduce breast cancer incidence.
Susceptibility to Breast Cancer: Dietary and Other Factors
Ralph J. Delfino, M.D., Ph.D.
University of California, Irvine
The proposed study will examine whether an elevated level of exposure to the food-borne carcinogens known as heterocyclic aromatic amines (HAAs) are associated with an increased risk of breast cancer in women. It is biologically plausible that such an association may exist given over 2 decades of experimental research on these potential carcinogens, including several studies showing that they are potent mammary gland cancer causing agents (carcinogens) in rodents. It is now an appropriate time to examine whether there is any risk of breast cancer in women attributable to exposure to dietary HAAs. These substances are formed when meat is cooked by many common methods at high temperatures, e.g., barbecuing. Simple methods are available to prevent their formation prior to any preferred method of cooking. Therefore, the proposed research could ultimately point to practical approaches to the primary prevention of breast cancer. In addition, the proposed study will examine a well-characterized gene known as NAT2, which is involved in the formation of deleterious mutations caused by HAAs. The proposed case-control study will recruit 2400 women who are scheduled for a diagnostic breast biopsy to rule out a carcinoma (the most common form of breast cancer), with an expected 600 cases of breast cancer and 1800 controls with benign breast diseases. Since subjects will be interviewed prior to the biopsy, selection and recall bias problems, which are common to case-control studies, will be reduced considerably. The control group will also be divided and compared based upon their biopsy results which are thought to indicate either high, low, or no risk of future breast cancer. An interview-administered questionnaire will be used to assess dietary exposures to HAAs. In addition, other important factors, which may influence or interfere with the exposures of interest or interact with them, will be assessed with self-administered dietary and risk factor questionnaires so that they may be taken into account. Diagnoses (from 4 participating breast cancer treatment centers) will be confirmed by the research pathologist. Known or suspected risk factors for breast cancer will be controlled for, including, dietary nutrients, body mass, temporal changes in body weight, height, age, menstrual, pregnancy and lactation history, family history of breast and ovarian cancer, alcohol intake, steroid hormone use, socioeconomic status and race/ethnicity.
In summary, the proposed study will extend research on the relationship between breast cancer and diet, by examining specific agents which may explain the inconsistency of other epidemiological studies showing that high levels of meat consumption, and possibly fat, are associated with breast cancer risk. The present research approach increases the relevance of experimental research in animals to specific human populations by linking genetically-determined metabolic susceptibility to genetic damage, along with assessments of the relevant exposures.
A better understanding of the importance of HAAs and NAT2 genotype can provide important insights into: (1) possible breast cancer-causing mechanisms; and (2) practical approaches to the primary prevention of breast cancer in the general population.
Alcohol and Risk of Estrogen-Receptor Positive Breast Cancer
Shelley M. Enger, Ph.D.
University of Southern California
In this postdoctoral fellowship, the association of alcohol intake with risk of breast cancer will be assessed separately for estrogen-receptor-positive and estrogen-receptor-negative tumors, using data from two large epidemiologic studies of risk factors for breast cancer. Estrogen receptors are cellular proteins that bind estrogen, a hormone that stimulates growth of breast tissue. Breast tumor cells with higher levels of estrogen receptors (estrogen-receptor-positive tumors) respond better to hormonal therapies than breast tumor cells with low levels of estrogen receptors (estrogen-receptor-negative tumors). If alcohol intake is related only to estrogen-receptor-positive tumors, this may clarify the role of alcohol in the etiology of breast cancer, and potentially support an estrogen-mediated effect of alcohol on breast cancer. In addition, an association of alcohol with risk of breast cancer that differs for estrogen-receptor-positive and estrogen-receptor- negative cancers would support the hypothesis that estrogen-receptor status defines different types of breast cancer, rather than two stages in the development of one disease.
Subjects are 2,330 women diagnosed for the first time with breast cancer (cases) and 2,330 women without breast cancer (controls) participating in two population-based case-control studies (Studies A and B) of risk factors for breast cancer. All subjects are white (Anglo or Hispanic) female residents of Los Angeles County. Each case subject is individually matched to a control subject on age, parity (Study A only), and neighborhood of residence. Eligible cases in Study A were all women in Los Angeles County aged 40 years or younger diagnosed with in situ (localized) or invasive breast cancer from July, 1983, through December, 1988. Eligible cases in Study B were all women in Los Angeles County aged 55-64 years diagnosed with in situ or invasive breast cancer from March, 1987, through December, 1989. Alcohol consumption, demographics, and other information was obtained in interview questionnaires. Estrogen-receptor status was determined for all case subjects, but for 80 percent of the subjects the original pathology laboratories must be queried for the results. Appropriate statistical analyses will be used to estimate the association of alcohol intake with risk of estrogen-receptor-positive and estrogen-receptor-negative breast cancer.
The results of this research may increase understanding of mechanisms through which alcohol might increase the risk of breast cancer, and the postdoctoral fellow will gain valuable experience working in a renowned breast cancer research program, leading to a career in breast cancer research.
Genetic Susceptibility to Breast Cancer
Brian E. Henderson, M.D.
University of Southern California
A substantial body of experimental, clinical and epidemiologic evidence indicates that hormones play a major role in the etiology of breast cancer. The known risk factors for breast cancer can be understood as measures of the cumulative exposure of the breast to estrogen and, perhaps, progesterone. Exposure to these hormones cause breast cells to grow and divide. We hypothesize that certain genes involved in the metabolism of estrogen in the body, such as those that convert estrogen from less active to more active forms, play important roles in breast cancer. We hypothesize that changes in the estrogen receptor (ER) gene, which works in concert with estrogen and other factors to signal breast cells to divide, are associated with breast cancer risk.
We propose to conduct a study to examine the possible associations between three genes and breast cancer in 400 women from an ethnically diverse cohort in Los Angeles County and Hawaii established in 1993. Fifty breast cancer cases and fifty controls between 45 and 74 years old from each of the following ethnic groups will be included in the study: African-American, Hispanic, Japanese, and white. We have also collected data on family history, diet, and other lifestyle habits from these women via a mailed questionnaire. The questionnaire was translated into Spanish for study participants who were not English Speaking.
To date, very few studies have looked at the possible genetic components of breast cancer between and within different ethnic populations, and no study has focused on these specific genes. The significance of this proposed study lies in its multi-disciplinary approach, focus on multiethnic pop- ulations and potential future impact. If this work identifies associations between these genes and breast cancer, it could be used to identify possible targets of intervention such as modification of estrogen metabolism within breast cells to slow or stop the proliferation of breast cancer cells.
Vitamin D & Genetics of Vitamin D Receptor in Breast Cancer
Sue A. Ingles, Ph.D.
University of Southern California
The specific aims of this proposal address the CBCRP priority issues of the underlying biological causes of breast cancer (etiology) and the development of the disease (pathogenesis): we plan to test whether genetic variants in the vitamin D receptor (VDR) are related to breast cancer incidence and whether blood vitamin D levels interact with the genetically determined VDR variants to influence the risk of breast cancer. These genetic variants have recently been shown to have a strong effect on bone density; and, since vitamin D plays an important role in cellular growth and differentiation (the process by human body cells become different and specific, e.g., a kidney or breast cell), it is possible that variation in the VDR may affect breast cancer in a similar manner.
To investigate this hypothesis, we propose to conduct a case control study of 200 matched pairs of breast cancer cases and controls (that is, similar women who do not have breast cancer), equally divided among four ethnic groups: African American, Hispanic, Japanese, and White. DNA from cases and controls will be typed (examined) for the commonly used marker (BSMI) which is tightly linked to the suspected VDR causal mutation. We will also type one of the variants which is a candidate for the causal mutation. Since inconsistencies among previous studies suggest that different BSMI variants may be associated with the causal mutation in different ethnic populations, we will examine linkage disequilibrium between the two loci in each of the four ethnic groups. These data will provide information about the usefulness of BSMI as a measure for the VDR causal mutation.
Given the role that vitamin D is known to play in cellular growth and differentiation, the recent work using vitamin D analogues to suppress the growth of breast cancer cells, and the recent discovery of VDR variants which appear to account for a large proportion of genetic variation in bone density, we feel that there is sufficient evidence to investigate the role of the VDR in breast cancer incidence. A positive result would have implications for breast cancer prevention and treatment: administration of vitamin D or its analogues might be useful for prevention of breast cancer or its progression in individuals with high risk variants. Since the suspected high risk variant is common in the population, a positive result could have a large impact on reducing the incidence of breast cancer in California.
Case-Control Study of Breast Cancer in Asian-American Women
Anna H.Wu, Ph.D.
University of Southern California
The main objective of this population-based case-control study of breast cancer in Asian Americans is to investigate the causes of their increased breast cancer rates compared to in their rates in Asia. The proposed three-year investigation will include interviews with 350 Asian-Americans (Chinese, Filipino, Japanese), ages 25 to 74, diagnosed with histologically-confirmed (i.e., confirmed by examination of biopsied breast tissue), primary invasive breast cancer identified in Los Angeles County. An equal number of Asian Americans without breast cancer, matched to cases on age, ethnicity, and neighborhood (the “control” group) will also be interviewed. A standardized in-person interview will be conducted and will include a comprehensive diet history covering usual diet during the year before diagnosis for breast cancer cases and the year before interview for controls.
The study design and sample size will allow us to test the main hypothesis that high intakes of soy products, vegetables and fruits, dietary fiber, and specific micronutrients reduce the risk of breast cancer and that high intakes of fat increase the risk of breast cancer. We will also examine the association of breast cancer with physical activity, body size, body fat distribution, and weight change.
While this study is a complete, self-standing study, we are very fortunate in being able to conduct this study at this time for two reasons. First, it will allow us to compare our results on physical activity and use of exogeneous hormones to those obtained in the Los Angeles component of the nationwide CARE study (which is not covering the Asian-American community or dietary factors). Second, this study will build upon, and expand our study on Asian-Americans conducted in the 1980s by adding the important 55-74 age group, information on physical activity, and specifics of dietary factors. We can begin to answer some of the very important new questions which have been raised in the area of physical activity, diet, and cancer during the last decade of research. Thus, with our relatively modest grant, we will be able to gain the advantages of much more costly research conducted with other funds.
In summary, this study will help to provide a better understanding of the role of soy and other modifiable risk factors (i.e., other diet, physical activity, body size) in the etiology of breast cancer. Achieving these objectives will have substantial public health significance not only for Asian-Americans, but for all racial/ethnic groups, and will have important implications in the prevention of this disease.
