Folate and Folic Acid Supplement Use and Breast Cancer Risk in BRCA1/2 Mutation Carriers

Transcription

1 Folate and Folic Acid Supplement Use and Breast Cancer Risk in BRCA1/2 Mutation Carriers by Shana Jean Kim A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Nutritional Sciences University of Toronto Copyright by Shana Jean Kim 2016

2 Folate and Folic Acid Supplement Use and Breast Cancer Risk in BRCA1/2 Mutation Carriers Shana Jean Kim Master of Science Department of Nutritional Sciences University of Toronto 2016 Abstract The relationship between folate (a B vitamin important in DNA synthesis and methylation reactions) and cancer risk among BRCA1/2 mutation carriers with a genetic predisposition for developing breast cancer is unclear. Therefore this study investigated the association between plasma folate and folic acid supplement use, along with other B vitamins, and breast cancer risk in BRCA1/2 mutation carriers. Women with high plasma folate concentrations had significantly increased risks for breast cancer, compared with women with low plasma folate concentrations. However, women who never used folic acid-containing supplements had significantly increased risks for breast cancer. Similarly, never use of vitamin B6- or vitamin B12-containing supplements tended to be associated with increased breast cancer risk. These findings support a U -shaped association of folate and breast cancer risk and suggests that moderate B vitamin supplement use may be protective for BRCA1/2-associated breast cancer, however BRCA1/2 mutation carriers should be cautious of over-supplementation. ii

3 Acknowledgments To my supervisor, Joanne. Your support and unwavering confidence in me has meant so much. I came into this project seeking an academic challenge, and you have certainly fulfilled that expectation. You have truly provided me with a unique opportunity to grow as a student, and this experience has shown me what I am capable of accomplishing. I am also so grateful for your down-to-earth, level-headedness throughout this experience, and for your personal interest in my endeavors beyond this thesis. Thank you for being my supervisor, mentor, and friend. To my advisory committee members, Dr. Young-In Kim and Dr. Mohammad Akbari. Thank you for your dedication and interest in my project. Your sound advice and expertise have helped shape my project into what it has become. I would also like to thank my appraiser, Dr. Michelle Cotterchio, for the thoughtful comments made on my thesis. I thank you all for your kind words at my defense. To everyone at the FBCRU, you have made working long hours at the office bearable! Thank you everyone who has helped me at one time or another, including Dina, Marcia, Sophia, Jenn, Pam, Javaid, Vasily, Rania, Nicole, Dawn, Aletta, Ping, Rob, Ellen, Farah, Cindy, Ravleen, Asrafi, and Yaminee. And a special thank you to Olivia, you have provided me with countless hours of entertainment and laughter, and I am proud to be your surrogate twin. To Vanessa and Effie, for keeping me sane these past two years. Your friendship has meant a lot to me. Thank you for the endless nights of beers, nachos, and music. Finally, to my parents. I am so thankful for all the opportunities you have given me. You taught me the value of hard work. I especially would like to dedicate this to my rock, who won her own battle against breast cancer, my mom. Thank you everyone for being a part of this journey with me. iii

4 Table of Contents Abstract... ii Acknowledgments... iii Table of Contents... iv List of Tables... vii List of Figures... viii List of Appendices... ix Chapter 1 Introduction...1 Chapter 2 Literature Review The Breast Cancer Susceptibility Gene-1 (BRCA1) and Breast Cancer Susceptibility Gene-2 (BRCA2) and Breast Cancer Overview of BRCA1/2-Associated Breast Cancer Mutation Classification Mechanism of Action of the BRCA1/2 proteins BRCA1/2-Associated Breast Carcinogenesis Breast Cancer Management for BRCA1/2 Mutation Carriers Screening Prevention Options for BRCA1/2 Mutation Carriers Modifiers of Risk in BRCA1/2 Mutation Carriers Hormonal and Reproductive Modifiers Lifestyle and Dietary Modifiers Folate Chemical Structure and Metabolism Biochemical Function Folate and Health Mandatory Folic Acid Fortification and Prenatal Supplementation...18 iv

5 2.2.4 Assessment of Folate Status Current Folate Status of the North American Population Folate and Breast Cancer Evidence in the General-Risk Population Animal Studies Observational Studies Clinical Trials Evidence in High-Risk Populations Mechanism of Action of Folate in Carcinogenesis Other B Vitamins and Breast Cancer Vitamin B6 Overview Vitamin B12 Overview Vitamin B6 and Vitamin B12 and Breast Cancer...38 Chapter 3 Rationale, Objectives, and Hypotheses Rationale Objectives Hypotheses...42 Chapter 4 Plasma Folate, Vitamin B6, and Vitamin B12 and Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers Abstract Introduction Methods Study Population Data and Sample Collection Study Subjects Available for Analysis Laboratory Assays...46 v

6 4.3.5 Statistical Analysis Results Discussion...50 Chapter 5 Folic Acid and Other B Vitamin Supplement Use and Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers Abstract Introduction Methods Study Population Data Collection Subject Selection Statistical Analyses Results Subject Characteristics Supplement Use in Adulthood and Breast Cancer Risk Supplement Use in Pregnancy and Breast Cancer Risk Effect Modification Discussion...66 Chapter 6 General Discussion General Discussion Strengths and Limitations Future Directions and Conclusions...78 References...80 Appendices...94 Copyright Acknowledgements...99 vi

7 List of Tables Table 2.1. Summary of convincing, probable, and likely modifiers of sporadic breast cancer risk among premenopausal and postmenopausal women compared with BRCA1/2-associated breast cancer risk Table 2.2. Folate concentrations in serum/plasma and RBC for determining folate status in all age groups, using macrocytic anaemia as a haematological indicator Table 2.3. A summary of all prospective and nested case-control studies investigating the relationship between circulating folate concentrations and breast cancer outcomes Table 2.4. A summary of all clinical trials investigating the relationship between folic acid supplement use and breast cancer outcomes Table 4.1. Baseline characteristics of the study population by plasma folate concentrations Table 4.2. HR and 95% CI of breast cancer by plasma folate, vitamin B6, and vitamin B Table 4.3. HR and 95% CI of breast cancer by plasma folate according to BRCA1/2 mutation status and menopausal status Table 5.1. Characteristics of BRCA1/2 mutation carriers included in the study by breast cancer cases and controls Table 5.2. OR and 95% CI of breast cancer risk by supplement use in BRCA1/2 mutation carriers Table 5.3. OR and 95% CI of breast cancer risk by supplement use in BRCA1/2 mutation carriers during pregnancy Table 5.4. OR and 95% CI of breast cancer risk by total daily folic acid, vitamin B6 and vitamin B12 supplement use stratified by BRCA1/2 mutation type vii

8 List of Figures Figure 2.1. Chemical structure of folate Figure 2.2. One-carbon metabolism cycle Figure 2.3. Forest plot of case-control studies investigating dietary folate intake and breast cancer risk Figure 2.4. Forest plot of prospective studies investigating daily dietary folate intake and breast cancer risk Figure 2.5. Forest plot of prospective studies investigating daily total dietary folate intake (including supplements) and breast cancer risk Figure 2.6. Summary of the dual modulatory role of folate in carcinogenesis Figure 2.7. Forest plot of serum PLP and total dietary vitamin B6 intake and breast cancer risk 39 Figure 2.8. Forest plot of serum vitamin B12 and total dietary vitamin B12 intake and breast cancer risk Figure 4.1. Flow diagram of the subject selection for the prospective evaluation of plasma folate and other B vitamins and breast cancer risk Figure 5.1. Flow diagram of the subject selection for the evaluation of supplement use and breast cancer risk viii

9 List of Appendices Appendix 1. HR and 95% CI of breast cancer by quintiles of plasma folate, vitamin B6, and vitamin B Appendix 2. Supplemental questionnaire ix

10 Chapter 1 Introduction Women who inherit a deleterious BRCA1/2 mutation face a high lifetime risk of developing breast cancer, estimated up to 87% compared with 12% in the general population (1, 2). Given the highly penetrant nature of these mutations, the need for evidence-based recommendations for breast cancer risk management is of extreme importance. Evidence suggests there is a role for non-genetic modifiers of risk in BRCA1/2 mutation carriers, such as reproductive and hormonal factors (3, 4), however the role of diet has yet to be clearly elucidated (5). Folate, a water-soluble B vitamin and an important co-factor in one-carbon metabolism, is wellpositioned to influence the pathogenesis of several chronic diseases including cancer as it plays essential roles in DNA synthesis and biological methylation reactions. Despite reports from casecontrol studies suggesting the possible cancer-protective effects of folate (6), some prospective studies suggest that high circulating and dietary folate may increase tumor progression and breast cancer risk (7, 8). This causes a reason for concern as there has been a dramatic increase in circulating folate concentrations in the population since the introduction of mandatory food fortification with folic acid (the synthetic form of folate) in North America in 1998 (9, 10), reports that 30 40% of the North American population are consuming folic acid-containing supplements on a daily basis (11-13), and recommendations that women of childbearing age consume folic acid supplements to help prevent neural tube defects (14). The potentially harmful effect of high folate status on breast cancer (8), the increase in folate and folic acid intake at the population level (9-13), the well-described dual effects of folate on carcinogenesis (15), and the heightened predisposition for cancer among BRCA1/2 mutation carriers underscores the need to clarify a role of this vitamin in the development of breast cancer (8). However no previous studies to our knowledge have examined the relationship between folate or folic acid supplement use and breast cancer risk in BRCA1/2 mutation carriers in North America, where fortification practices exist. Therefore the objective of this thesis was to investigate the relationship of folate and folic acid supplement use and breast cancer risk among BRCA1/2 mutation carriers. Additionally, other B 1

11 2 vitamins of importance, such as vitamin B6 and vitamin B12, were examined as they are also important co-factors in one-carbon metabolism and are commonly found in similar foods as folate. This work will help to develop evidence-based recommendations for women with a high genetic predisposition for developing breast cancer and may have a significant impact on the lives of these women.

12 Chapter 2 Literature Review 2.1 The Breast Cancer Susceptibility Gene-1 (BRCA1) and Breast Cancer Susceptibility Gene-2 (BRCA2) and Breast Cancer Overview of BRCA1/2-Associated Breast Cancer Breast cancer is the most common cancer afflicting women worldwide (16). In Canada, a women s lifetime risk of developing breast cancer is estimated to be 12%, with an estimated 25,000 new breast cancer cases in 2015 (2). Although the incidence of breast cancer in women is high, there has been a drastic decline of death from breast cancer over the last 30 years in Canada, with an observed drop in age-standardized mortality rates by 44% since 1986 (2). This observed decline is likely attributed to increased screening, early-detection programs, and modern advances in cancer treatment therapies (2, 16). Despite the reduced probability of dying from breast cancer in Canada, this disease remains the second leading cause of cancer-related death in female Canadians with the approximated lifetime risk of dying from breast cancer being 3% (2). Several factors influence an individual s risk for developing breast cancer, an important contributor of which is a family history of the disease (17). This highlights the importance of genetic factors as determinants of risk and it has been estimated that up to 10% of all breast cancers are hereditary cases, i.e., attributed to known inherited germline mutations (18). The best described and most substantial contributors to these hereditary breast cancers are attributed to the deleterious mutations in the BRCA1 and BRCA2 genes. The breast cancer susceptibility genes, BRCA1 and BRCA2, are tumor suppressor genes located on chromosome 17q21 and 13q12, respectively (19, 20). BRCA1 was first discovered through linkage analysis in families with several cases of early-onset breast cancers in the early 1990 s, and later identified by positional cloning in 1994 (19, 20). Soon after, BRCA2 was identified and cloned in 1995 (19, 20). 3

13 4 Although the prevalence of BRCA1 and BRCA2 mutations in the general population have been estimated to be 0.4%, inherited BRCA1/2 mutations are highly penetrant genes and are associated with an increased lifetime risk of developing breast, ovarian, and certain other cancers compared to the general population (19, 21). Women with a BRCA1/2 mutation have previously been reported to have a lifetime breast cancer risk ranging from 40 87%, and an estimated average lifetime risk of 65% (1, 22). Women with a BRCA2 mutation have reported lifetime risks of breast cancer ranging from 27 84%, with average lifetime risks of 45% (1, 22). Male BRCA1/2 mutation carriers are also associated with increased risks of developing breast cancer, with lifetime risks of developing male breast cancer ranging from 1 5% in BRCA1 mutation carriers, and more substantially, 5 10% in BRCA2 mutation carriers, compared with 0.1% in the general population (23). Carriers of the BRCA1/2 mutation are also associated with increased risks of ovarian cancer, prostate cancer, pancreatic cancer, and melanoma (24). Women with BRCA1/2 mutations are more likely to be diagnosed with breast cancers at an early age (typically before the age of 50) (25), have an elevated risk of developing ipsilateral or contralateral breast cancer (26, 27), and have higher grade tumors compared with sporadic cancers (28). BRCA1-associated breast cancers typically exhibit pathologically aggressive tumors that are estrogen receptor- (ER), progesterone receptor- (PR), and Her2/neu-negative (29). BRCA2-associated tumors however typically do not display distinct pathology from sporadic breast cancers of the general population and tend to be ER-, PR-positive, and Her2/neu-negative (19, 29). BRCA1 mutation carriers are typically associated with worse overall breast cancer survival compared to non-carriers, however BRCA2 mutation carriers are not significantly different from non-carriers in terms of overall breast cancer survival (30) Mutation Classification Several sequence variations for the BRCA1 and BRCA2 mutation have been identified, which are classified as pathogenic mutations, benign variants, or variants of unknown significance (VUS) (31). The majority of pathogenic mutations in BRCA1 and BRCA2 are frameshift mutations which result in protein truncation (21). However missense mutations, large rearrangements due to large deletions of exons, and insertions/duplications also contribute to pathogenic mutations and protein truncation (21). Evidence has also shown that breast and ovarian cancer risks among

14 5 BRCA1/2 mutation carriers can vary based on the type and location of the mutation (32). Benign variants of the BRCA1/2 gene include polymorphisms observed in the general population which are not associated with increased risk and do not significantly affect protein function (31). A VUS has an unknown effect on protein function and breast or ovarian cancer risk, and are typically missense mutations where a single nucleotide change results in an altered amino acid (31). Specific pathogenic mutations arise more frequently in certain populations because of a shared common ancestry, and these variants are known as founder mutations. For example, three founder mutations have been identified in those with an Ashkenazi Jewish ancestry (BRCA1 185delAG and 5382insC; BRCA2 6174delT) and it is very unlikely that an Ashkenazi woman would carry a different mutation outside of these founder mutations (19). Other founder mutations have been identified in Polish, Icelandic, Norwegian, French Canadian, Chinese, and Japanese populations, to name a few (33) Mechanism of Action of the BRCA1/2 proteins The BRCA1 and BRCA2 proteins function in several roles to maintain genomic stability, DNA repair, cell proliferation, and cell differentiation. Both BRCA1 and BRCA2 predominantly act together in a common pathway to repair DNA double-stranded breaks (DSBs) with homologous recombination (19, 34). Using the sister chromatid as a template for repair, BRCA1, PALB2 and, BRCA2 proteins localize RAD51, a protein needed for error-free DSB repair (19, 35). In the absence of BRCA1 or BRCA2, the cell uses error-prone methods that do not use the sister chromatid as a template, such as non-homologous end joining (19). This alternative method of repair can result in an increased likelihood of accumulating mutations and chromosomal instability, which can ultimately lead to carcinogenesis. In addition, BRCA1/2 deficient cells are more susceptible to DSBs upon exposure to agents such as cisplatin, mitomycin C or ionizing radiation, predisposing the cell to carcinogenesis (19). Other key functions of the BRCA1 protein include a role in checkpoint control, ubiquitination, chromatin remodeling, repair of stalled/collapsed replication forks, transcriptional regulation, X- chromosome inactivation, oxidative stress response, hormonal signaling, and differentiation of breast epithelial cells (19, 25, 36).

15 BRCA1/2-Associated Breast Carcinogenesis The breast is comprised of glandular tissue (composed of ducts and lobules), supporting connective tissue, fibrous tissue, and neurovascular bundles (37). Breast carcinomas typically originate from the epithelial layer of the glandular tissue and are hypothesized to progress in stages from a normal cell to in situ carcinoma, invasive carcinoma, then metastatic carcinoma (37). Hyperplasia and hyperproliferation may lead to carcinoma in situ (37). Two types of in situ carcinomas can originate from the ductal cells or lobule cells, and can progress to invasive carcinoma, where the cancerous cells invade the basement membrane of the epithelium and spread to surrounding tissues. Metastatic carcinoma would then be the invasion onto distant sites. Breast carcinogenesis likely originates due to a combination of genetic and epigenetic changes in the cell (38). The progressive accumulation of these changes then drive tumor progression. Genetic and epigenetic changes that result in tumor suppressor gene silencing have been demonstrated to enhance proliferation of cells and may result in carcinogenesis (38). Activation of proto-oncogenes have been linked to increased cellular proliferation and breast cancer development, some oncogenes of which are HER2/neu, c-myc and ras (38). Furthermore, functional silencing of tumor suppressors such as p53, BRCA1, BRCA2, and PTEN similarly have been demonstrated to increase cellular proliferation and breast cancer development (38). Interestingly, BRCA1 silencing in sporadic breast tumors exhibit similar tumor characteristics as BRCA1/2-associated breast cancers and are typically triple-negative, early-onset, and have higher histological grade (39, 40). The BRCA1/2 mutations are inherited through an autosomal dominant fashion and the inheritance of one functional copy and one mutated copy of the BRCA1/2 gene results in haploinsufficiency, or decreased BRCA1/2 protein expression (41). Consequently, this can reduce the cells ability to undergo error-free DSB repair under various cellular and environmental stresses as there is insufficient amount of protein available, and this heterozygous state can lead to genomic instability. However, it has also been suggested that in the haploinsufficient state, further loss of the single wild type allele in BRCA1/2 mutation carriers (through a somatic mutation or less likely through epigenetic silencing) is required for BRCA1- mediated tumourigenesis (19). This is known as loss of heterozygosity (LOH) in the two-hit model of tumourigenesis hypothesis. This hypothesis states the inactivation of both alleles of a

16 7 tumour suppressor gene are required for tumourigenesis (19). In sporadic breast cancers, loss of both alleles would be required to initiate tumourigenesis however in BRCA1/2 mutation carriers, only one hit would be required, which may explain why BRCA1/2 mutation carriers experience early-onset breast cancer. Studies examining LOH in breast and ovarian tumours among BRCA1 mutation carriers reported that between % of the tumours had LOH strongly supporting the two-hit model hypothesis (42, 43). The breast stem cell theory interestingly proposes that breast cancer may derive from stem or progenitor cells that are dysregulated in their process of self-renewal (44). Therefore the larger the stem cell pool, the more likely breast cancer will occur (44). BRCA1 proteins have functional roles in stem cell regulation and have been shown to be involved in the differentiation of ERnegative stem cells to ER luminal cells (44). Women with BRCA1 mutations were also shown to have higher stem cell marker expression in only LOH lobules, compared to women of the general population (44). This suggests that loss of BRCA1 may result in increased stem cell pools due to inadequate stem cell differentiation, increasing genetic instability and increasing potential origins for breast cancer development. Additionally, there is an observed commonality that mutations in the BRCA1/2 genes predispose women to tissue-specific cancers of the breast and ovaries. This suggests that sex hormones may influence the development of BRCA1/2-associated cancers. Estrogen can induce single and double stranded DNA breaks likely through its production of oxidative metabolites that cause free radical-mediated DNA damage and through its action in estrogen-induced excessive proliferation which can result in an accumulation of DNA damage products (34, 45, 46). The lack of sufficient BRCA1/2 protein synthesis due to an inherited mutation or LOH could result in genomic instability and increased DNA damage in tissues with high levels of estrogen exposure such as the breast. Furthermore, progesterone-mediated signaling through receptor activator of nuclear factor κb (RANK) and RANK ligand (RANKL) has been linked with the development of BRCA1/2- associated breast cancer. Specifically, progesterone stimulates the binding of RANKL to RANK which increases mammary epithelial cell maturation and proliferation. As such, overexposure to progesterone enhances mammary tumor formation (47-50). Inhibition of RANKL and progesterone by pharmacological agents significantly suppresses mammary tumourigenesis in

17 8 BRCA1 deficient mice (51-53). Moreover, Widschwendter et al. reported significantly lower mean circulating levels of osteoprotegerin (OPG), the endogenous decoy receptor for RANKL that antagonizes RANK/RANKL-mediated signaling, as well as higher progesterone levels among premenopausal BRCA1 mutation carriers compared to non-carrier controls (54, 55). Increased progesterone concentrations are also implicated in the expansion of mammary stem and progenitor cell population which is of particular importance for BRCA1/2 mutation carriers given their propensity to develop breast and ovarian tumors with stem cell-like properties (48-50). Therefore the influence of sex hormones such as estrogen and progesterone may explain the tissue-specificity of the development of BRCA1/2-associated cancers Breast Cancer Management for BRCA1/2 Mutation Carriers Predictive genetic testing permits the identification of BRCA1/2 mutation carriers, allowing for these high-risk women to undergo tailored screening, primary prevention, and risk modification options for the management of their breast and ovarian cancer Screening Hereditary breast cancer screening guidelines set by the National Comprehensive Cancer Network (NCCN) recommend that clinical breast exams occur every 6 12 months beginning at age 25, and annual mammograms and breast MRIs begin at the same age (56). In addition, breast self-exams may be performed once a month, however this has not been shown to reduce breast cancer mortality (57) Prevention Options for BRCA1/2 Mutation Carriers Current primary prevention options for these high-risk women are limited to chemoprevention treatment with tamoxifen or invasive prophylactic surgery (25). Tamoxifen, a selective ER antagonist, can be effective in reducing ER-positive breast cancer in the general population (25). The evidence for primary prevention of breast cancer in BRCA1/2 mutation carriers with tamoxifen however is extremely limited as only a select number of women elect for tamoxifen chemoprevention treatment (4, 25, 58). Only one small study in 19 unaffected BRCA1/2 mutation carriers suggested that tamoxifen use may prevent breast cancer incidence in BRCA2 mutation carriers but not in BRCA1 mutation carriers (59). Alternatively, tamoxifen has been well supported to effectively prevent contralateral breast cancers in BRCA1/2 mutation

18 9 carriers. In a recent meta-analysis, tamoxifen use has been associated with an 53% reduced risk of contralateral breast cancer in BRCA1 and 61% reduced risk in BRCA2 mutation carriers (60). Based on the evidence, tamoxifen can offer significant secondary prevention for the development of contralateral breast cancer, which BRCA1/2 mutation carriers are at an increased risk, however its effectiveness in primary prevention of breast cancer is still uncertain. Furthermore, tamoxifen can only offer transient protection during the course of treatment (typically five years) and several years after, and does not offer the lifetime protection that is offered by preventative prophylactic surgical means (25). Prophylactic bilateral mastectomy is currently the most effective method of primary prevention for BRCA1/2 mutation carriers as this confers lifelong protection of breast cancer by 90 95% (61). Although the level of protection is high, the rate of uptake of bilateral mastectomy among BRCA1/2 mutation carriers can be low and varies by country due to fear of disfigurement and side effects, with only 18% of BRCA1/2 mutation carriers electing to undergo prophylactic mastectomy (58). Another suggested method of primary prevention for BRCA1/2 mutation carriers is prophylactic bilateral salpingo-oophorectomy to remove the ovaries and the fallopian tubes. Along with impacting breast cancer incidence, oophorectomy has shown to significantly prevent cancers of the ovary, fallopian tube and peritoneum (25, 62). Bilateral salpingo-oophorectomy was previously believed to reduce the risk of breast cancer by 51% if completed before the age of 40 (as most BRCA1/2-associated breast cancers occur prior to the age of 40) (63); however the protective role of preventive bilateral salpingo-oophorectomy on breast cancer risk among women with a BRCA1 or BRCA2 mutation has recently been under debate as the majority of these protective findings were limited by several inherent biases which were not accounted for in the analyses (64). A prospective study from the Netherlands adjusting for these biases conferred by the previous biased studies ultimately showed no effect of prophylactic bilateral salpingooophorectomy on breast cancer risk, which was further confirmed with a prospective study by Kotsopoulos et al. (64, 65). Although the effectiveness of prophylactic bilateral salpingooophorectomy on preventing breast cancer incidence is currently under dispute, BRCA1/2 mutation carriers are still recommended for prophylactic bilateral salpingo-oophorectomy as it can significantly reduce their risk of ovarian cancer.

19 Modifiers of Risk in BRCA1/2 Mutation Carriers Although current prevention options such as chemoprevention with tamoxifen and invasive surgery are available for BRCA1/2 mutation carriers, the uptake of these choices is low due to fear of disfigurement and side effects. Thus alternative, nonsurgical prevention options including diet and lifestyle modifications are desired by this population. As previously reported, the estimated lifetime risk for developing breast cancer among BRCA1 and BRCA2 mutation carriers is quite wide, ranging from 40 87% and 27 84% respectively (22). Although genetic factors such as allelic variation of the mutation and presence of other modifying genes may vary breast cancer risk among BRCA1/2 mutation carriers, there is an observed variance of breast cancer risk even among family members who carry the same mutation (4, 22). Furthermore, lifetime breast cancer risks are higher in BRCA1/2 mutation carriers in recent decades compared to those born in earlier decades (4, 22). This observed incomplete penetrance and variance in breast cancer risk among individuals carrying a BRCA1/2 mutation suggests there is potential for risk modification and that there is a role for non-genetic determinants of risk including reproductive, dietary, and lifestyle factors (4). The dietary, nutrition, and physical activity factors for the prevention of breast cancer in the general population determined by the World Cancer Research Fund and American Institute for Cancer Research (AICR) in the breast cancer 2010 report is summarized in Table 2.1 and compared with the evidence for BRCA1/2 mutation carriers (66). Although previous studies have explored non-genetic modifiers of risk in the general population, these findings are not necessarily applicable for BRCA1/2 mutation carriers. Therefore there is a clear need to identify evidence based modifiers of penetrance to manage breast cancer risk among BRCA1/2 mutation carriers specifically, and the next section will review the current hormonal, reproductive, lifestyle and dietary factors that may influence risk in these high-risk women.

20 11 Table 2.1. Summary of convincing, probable, and likely modifiers of sporadic breast cancer risk among premenopausal and postmenopausal women compared with BRCA1/2- associated breast cancer risk. Risk Modifier Sporadic Breast Cancer Risk BRCA1/2-associated Premenopausal Postmenopausal Breast Cancer Risk Lactation/breastfeeding Alcohol Body fatness Abdominal fatness?? Total fat?? Adult weight gain?? Physical activity Adult attained height? Greater birth weight?? Legend: = decreased breast cancer risk; = increased breast cancer risk; = no association with breast cancer risk;? = unknown association with breast cancer risk Hormonal and Reproductive Modifiers The association between several hormonal and reproductive factors and breast cancer risk have previously been investigated as BRCA1/2 mutations are strongly associated with cancers of the breast and ovary. Interestingly, most hormonal and reproductive risk factors that influence breast cancer risk in the general population are also consistent in BRCA1 mutation carriers. For example, earlier age at menarche (<11 years old) is associated with increased breast cancer risk in BRCA1 mutation carriers compared with later age at menarche ( 14 years) (67). This finding is also observed in the general population (67). Furthermore, longer duration of breastfeeding is inversely associated with breast cancer among BRCA1 mutation carriers. Generally, breastfeeding > 1 year is associated with a 32 50% decrease in breast cancer risk compared with women who never breastfed (68, 69). Again this observation is consistent with the general population. Additionally, oral contraceptive use has been shown to increase breast cancer risk in BRCA1 mutation carriers (70), but shown to be protective for ovarian cancer (71). While parity is clearly a protective factor among women in the general population, the association between parity and breast cancer among BRCA1/2 mutation carriers is not clear. When comparing parous and nulliparous BRCA1 mutation carriers, there was no observed protection of childbirth and breast cancer risk (4, 69). Only when investigating the number of births was there a beneficial effect of having four or more births and breast cancer risk BRCA1

21 12 mutation carriers (OR 0.62; 95% CI 0.41, 0.94; P = 0.02) (72). In contrast, increasing parity among BRCA2 mutation carriers was associated with increased breast cancer risk (72). This observed difference between the general population and BRCA1/2 mutation carriers could be due to the reason that the breast tissue may fail to significantly differentiate during and after pregnancy in the absence of the BRCA1/2 proteins (73) Lifestyle and Dietary Modifiers Other modifiable determinants of risk include lifestyle and dietary factors, although the findings from these studies are much less clear and investigated to a lesser extent compared to hormonal and reproductive modifiers of risk. Although the AICR determined that there is convincing evidence that alcohol intake increases breast cancer risk in the general population (66), alcohol intake in any form (wine, beer, spirits) was not associated with breast cancer risk in either case-control or prospective studies in BRCA1/2 mutation carriers (74, 75). In the largest prospective investigation of 3,067 unaffected BRCA1/2 mutation carriers, women who ever consumed alcohol were not associated with breast cancer risk (RR 1.06; 95% CI 0.78, 1.44; P = 0.73) compared with women who never consumed alcohol. Cumulative alcohol use and age at first alcohol use were additionally not associated with breast cancer risk (P-trend = 0.65 and 0.76, respectively) (74). For factors related to body weight, the AICR established that body fatness decreases premenopausal breast cancer risk, while this and other measures of body fat (such as abdominal fatness, adult weight gain, and total fat) increases postmenopausal breast cancer risk (66). The mechanism as to why body fatness is protective for premenopausal breast cancer is currently unknown (66). These findings are generally consistent among BRCA1/2 mutation carriers as findings from larger case control studies show evidence that maintenance of a healthy body weight, both in early adult life and later on in life, may protect against BRCA1/2-associated breast cancer (76). In the largest study to date, weight loss of at least 10 lbs from the age of 18 to 30 was associated with significantly decreased BRCA1/2-associated breast cancer risk, while weight gain during the same period was not associated with overall risk (77). Further examination of subgroups determined that adult weight gain in BRCA1 mutation carriers with at least two children was significantly associated with increased breast cancer risk, however given the reduced sample size in this analysis, further investigation on weight gain and BRCA1/2-

22 13 associated breast cancer risk is warranted (77). There are, however, no studies on abdominal fatness or total fat and breast cancer risk among BRCA1/2 mutation carriers to our knowledge. Additionally, the AICR also suggested that physical activity decreases risk for pre- and postmenopausal breast cancer (66). Similarly, there is evidence that increased physical activity in adolescence or early adulthood is protective for breast cancer risk among BRCA1/2 mutation carriers (76). Due to a lack of studies available, the relationship between adult attained height and greater birth weight in BRCA1/2 mutation carriers is unknown. Other risk modifiers that have been investigated among BRCA1/2 mutation carriers suggest there may be other non-genetic modifiers of risk for high-risk women. Although the evidence is inconsistent, a past history of smoking has been associated with an increase in breast cancer risk among BRCA1/2 mutation carriers (78). Coffee consumption has also been shown to be associated with breast cancer risk reduction in BRCA1/2 mutation carriers. In a dose-responsive manner, women who consumed increasing cups of caffeinated coffee had a significantly lower OR of developing breast cancer compared to never drinkers (P-trend = 0.02) (79). Another study found that high levels of caffeine and caffeinated coffee intake were associated with improved DNA repair capacity in BRCA1 mutation carriers (80). Furthermore, single nutrients such as selenium and iron may be associated in reducing breast cancer risk among BRCA1/2 mutation carriers. Oral selenium supplementation for three months reduced the number of chromosome breaks in BRCA1/2 mutation carriers to a normal level (81), and women in the highest tertile of plasma iron had a 57% lower risk of breast cancer compared to those in the lowest tertile (OR 0.43; 95 % CI 0.18, 1.04; P-trend = 0.06). This suggests that diet, including certain nutrients, may be a potential modifier of risk in BRCA1/2 mutation carriers. 2.2 Folate Folate, a water-soluble B vitamin, may be an important modifier of breast cancer risk as it plays a pivotal role in DNA synthesis and biological methylation reactions, aberrancies of which have all been implicated in carcinogenesis.

23 Chemical Structure and Metabolism Folate is the generic term that encompasses the different forms of folate conjugates, depending on the variations of the three structural components that make up the chemical structure of folate. Structurally, folate is made up of a pteridine ring, p-aminobenzoyl acid, and a glutamate moiety (Figure 2.1) (82). Different forms exist based on the oxidation state of the pteridine ring, the one-carbon groups attached to the pteroic acid at the N-5 and N-10 position, and the number of glutamate residues (82, 83). All naturally occurring folates found in food have a reduced pteridine ring while folic acid, the supplemental form of folate, has an oxidized pteridine ring (83). Figure 2.1. Chemical structure of folate Footnote to Figure 2.1: Structurally, folate is made up of three components: a pteridine ring, p- aminobenzoyl acid, and a glutamate moiety. Reproduced from Glynn SA, Albanes D. Folate and cancer: a review of the literature. Nutr Cancer. 1994;22(2): by permission from Taylor & Francis. Folate found naturally in foods are less chemically stable than folic acid and can lose their functional activity within days or weeks (84). Folic acid however is stable for months or years as it is more resistant to chemical oxidation (84). Folic acid is also much more bioavailable compared with natural folate (84). The bioavailability of natural folate is highly dependent on the methods of harvesting, processing, and preparation of the foods (84). Bioavailability of natural folate is also dependent

24 15 on the host s ability to absorb folate in the small intestine, which requires the removal of the polyglutamate chain (15). The folate polyglutamyl chain is removed by the enzyme glutamate carboxypeptidase II (GCPII) allowing for the absorption of the cleaved folate monoglutamate (15). Depending on the efficiency of this cleaving process in the host, this can reduce the bioavailability of natural folate by as much as 25 50% (84). The folate monoglutamate can be transported through four methods, one of which is a reducedfolate carrier (RFC) (15, 83). This transmembrane carrier is an anion exchanger that mediates folate delivery into a variety of cells including the liver (15, 83). RFC has a higher affinity for reduced folate particularly the folate monogulamate, 5-methyltetrahydrofolate (mthf), than folic acid however it may not be responsible for the majority of folate transport across tissues (15, 83). Another transmembrane carrier, the proton-coupled folate transporter (PCFT), contributes to folate absorption in low-ph environments such as in the small intestine, where folate absorption occurs (83). PCFTs have similar affinities for mthf and folic acid, and is primarily responsible for folate transport in the small intestine (83). A third mechanism of uptake is through a family of folate receptors (FR), which are anchored to cell membranes and transport folate through an endocytic process (15, 83). These receptors, although slower than the transmembrane carriers, have a very high affinity for folic acid and a lesser, but still high, affinity for mthf (83). Interestingly, the FRα isoform is primarily involved in folate transport in epithelial membranes; it is moderately expressed in certain normal epithelial cells and is markedly elevated in several carcinomas, including breast and ovarian tumors (83). Lastly, multidrug resistance-associated proteins (MRPs) are a family of transmembrane carriers that can export folate from tissues (83). MRPs have very low affinity for folate however they have a high capacity for folate transport (83). Although RFCs and PCFTs are expressed on the apical membrane of the intestinal mucosa, MRPs are likely responsible for folate transport through the basolateral membrane of the intestinal mucosa into portal circulation (83). Most of the folate is then transported into the liver, where it can either be stored or released into circulation for transport into other tissues (83). Folate monoglutamates found in circulation are primarily mthf (15, 83). Once taken into cells, folate is converted into polyglutamates by folylpolyglumate synthase (FPGS) in tissues, red blood cells (RBCs) and urine for retention so it cannot be transported back across the cell membrane (83). Polygutamates also act as better substrates for folate-dependent enzymes in one-

25 16 carbon transfer reactions in the cell (83). Glutamates are then removed by gamma-glutamyl hydrolase (GGH) to enter back into circulation when required (83) Biochemical Function The primary functional role of folate is mediation of the transfer of one-carbon units involved in nucleotide biosynthesis, and biological methylation reactions (15). Although the primary circulating form of folate is mthf, folic acid gets converted by dihydrofolate reductase (DHFR) and enters the one-carbon metabolism cycle as tetrahydrofolate (THF) (85). The methionine cycle, which mediates biological methylation reactions, is depicted on the top right of Figure 2.2. The mthf transfers its methyl group to homocysteine (hcys) to synthesize methionine (MET) and THF (15, 85). This process is catalyzed with methionine synthase (MS) which is dependent on vitamin B12, another important vitamin required in one-carbon metabolism (15, 85). This process generates methionine and ultimately, S-adenosylmethionine (SAM), the primary methyl group donor for biological methylation reactions in DNA, RNA, and histone methylation (15, 85). The folate cycle, which mediates nucleotide biosynthesis, is depicted on the left of Figure 2.2. THF generated from the hcys to methionine reaction gets converted to 5, 10-methyleneTHF (methf) by serine hydroxymethyl transferase (SHMT), a vitamin B6 dependent reaction (15, 85). SHMT acts to facilitate a reversible reaction of serine to glycine, an entry point of onecarbon units in this pathway (15). MeTHF can then be catalyzed by the irreversible conversion to mthf through methylenetetrahydrofolate reductase (MTHFR), or methf can be directed towards thymidylate and purine biosynthesis (15, 85). Thymidylate synthase (TS) uses methf to convert deoxyuridine-5-monophosphate (dump) to deoxythymidine-5-monophosphate (dtmp; thymidylate), a precursor for DNA synthesis (not shown in Figure 2.2) (15). methf can also be directed to de novo purine biosynthesis by being converted to 10-formyltetrahydrofolate (F-THF) and catalyzed to incorporate the formyl group of the F-THF to the C-2 and C-8 position of the purine ring by phosphoribosylglycinamide formyltranderase (GARFT) and phosphoribosylaminoimidazole carbozamide formyltransferase (AICARFT) (not shown in figure 2.2) (86).

26 17 Not surprisingly, genetic variants of enzymes involved in the folate metabolic cycle can affect circulating folate concentrations. Reduced in enzyme activity in the MTHFR 677C>T polymorphism has strongly been associated decreased concentrations of circulating folate, and decreased levels of mthf in blood cells (15). Homozygous carriers of the 677T variant have only 30% of normal enzyme activity, while heterozygotes have 65% normal enzyme activity. Figure 2.2. One-carbon metabolism cycle Footnote to Figure 2.2: In the folate cycle, folic acid is imported into cells and reduced to tetrahydrofolate (THF). THF is converted to 5,10-methylene-THF (me-thf) by serine hydroxymethyl transferase (SHMT). Vitamin B6 seems to have an influence on this reaction. me-thf is then either reduced to 5- methyltetrahydrofolate (mthf) by methylenetetrahydrofolate reductase (MTHFR) or converted to 10- formyltetrahydrofolate (F-THF) through a sequence of steps. mthf is demethylated to complete the folate cycle. With the demethylation of mthf, the carbon is donated into the methionine cycle through the methylation of homocysteine (hcys) by methionine synthase and its cofactor vitamin B12 (B12). The methionine cycle begins with homocysteine that accepts the carbon from the folate pool through mthf to generate methionine (MET). Methionine, through methionine adenyltransferase (MAT), is used to generate S-adenosylmethionine (SAM), which is demethylated to form S-adenosylhomocysteine (SAH). After deadenylation by S-adenosyl homocysteine hydrolase (SAHH), SAH is converted back to homocysteine, resulting in a full turn of the methionine cycle. Dashed arrows denote multiple biochemical steps. BHMT, betaine hydroxymethyltransferase; DHFR, dihydrofolate reductase; DMG, dimethylglycine; GLDC, glycine decarboxylase; TS, thymidylate synthase. Reproduced from Locasale JW. Serine, glycine and one-carbon units: cancer metabolism in full circle. Nat Rev Cancer. 2013;13(8): by permission from Macmillan Publishers Ltd: Nature Reviews Cancer.

27 Folate and Health Folate is found naturally in a variety of foods such as leafy green vegetables, broccoli, citrus fruits, legumes, yeast, and liver. Folic acid is fortified in the food supply in North America in foods such as enriched bread, pasta, flour, breakfast cereal, and rice. Since folic acid is more chemically stable and bioavailable than naturally occurring folate, the dietary folate equivalent (DFE) conversion was developed to standardize all forms of folate to reflect the differences in bioavailability. In this conversion, 1 DFE = 1 μg food folate = 0.6 μg folic acid from supplements and fortified foods consumed with foods = 0.5 μg folic acid from supplements taken on an empty stomach (87). The recommended dietary allowance (RDA) of folate to meet the requirements of nearly all healthy individuals is 400 µg DFE/day for adult males and females (87). The RDA is higher for women who are pregnant or lactating (600 µg DFE/day or 500 µg DFE/day, respectively) (87). The recommended upper limit (UL) is based on the intake of folic acid only and is set at 1000 g DFE/day (87). The UL was set by the Institute of Medicine as 1/5 th of the lowest dose observed to mask vitamin B12 deficiency (i.e g DFE/day) (87). A UL for natural folate from foods was not set because no adverse effects have been reported from high folate from food (87). Although folate deficiency in the North American population is uncommon, it can lead to adverse outcomes such as anemia, elevated homocysteine concentrations, neuropsychiatric disorders, neural tube defects (NTDs), other adverse pregnancy outcomes, and cancer development (87). High folate intakes however have been a reason for concern as it has been observed to interact with certain anti-seizure and anti-malaria medication, decrease natural killer cell cytotoxicity, mask vitamin B12 deficiency, induce epigenetic changes, and promote cancer growth (8). A significant contributor to the recent rise in folate intakes in North Americans is due to the mandatory fortification of the food supply with folic acid since 1998 and high folic acidcontaining supplement use Mandatory Folic Acid Fortification and Prenatal Supplementation With several studies providing evidence that folic acid use may reduce NTD, including evidence from randomized controlled trials (88, 89), the U.S. Public Health Service began to recommend that all women of childbearing age consume 400 µg of folic acid daily to prevent NTD starting in

28 (14). This triggered the implementation of fortification of the food supply with folic acid. In the United States, mandatory fortification of all white flour, enriched pasta and cornmeal with folic acid was authorized in 1996, then fully implemented in 1998 (14). Similarly in Canada, voluntary fortification programs to enrich grain products with folic acid began in 1996 (14), and mandatory fortification of 150 μg of folic acid/100 g of all white flour, enriched pasta and cornmeal was fully implemented in 1998 (14). In the same year, the IOM also suggested all women capable of becoming pregnant should consume 400 µg/day of folic acid from supplements or fortified foods to prevent NTDs (14). Although the mechanism of folate in reducing NTD is currently unclear, there have been significant declines in NTDs from 19 55% since the initiation of folic acid food fortification in Canada and other countries (14). The Centers for Disease Control and Prevention (CDC), Health Canada and the World Health Organization (WHO) strongly recommend supplementation of at least 400 μg of folic acid per day two to three months prior to pregnancy, during pregnancy and in the postpartum period (90-92). Additionally, they recommend all women of childbearing age to consume daily folic acid supplements, as many pregnancies may be unplanned (90-92). These recommendations are consistent with those recently set by the Society of Obstetricians and Gynecologists of Canada (SOGC) in 2015 for women at low risk for a NTD (93). However, the SOGC advise women at moderate risk for a NTD to supplement daily with 1000 ug of folic acid three months prior to conception until 12 weeks of gestation, and 400 or 1000 ug of folic acid daily in the postpartum period (93). High-risk women are advised to consume 4000 ug of folic acid daily from three months prior to conception until 12 weeks of gestation, then 400 or 1000 ug of folic acid daily in the postpartum period (93). Given that high dose folic acid supplements are recommended during the pre-/peri-natal period, pregnancy may be an important period to examine the potentially modifying effect of folic acid supplement use and breast cancer risk. Hormonal changes during pregnancy may provide a protective window in which the breast cells are less likely to transform due to increased mammary gland differentiation (94). However animal studies have shown that if the breast tissue has been exposed to damaging carcinogens prior to pregnancy, there is increased incidence and growth of mammary tumors due to increased breast cell proliferation upon exposure to pregnancy-related hormonal changes (94). Whether or not folic acid supplement exposure during this time can modify risk is not clear.