BREAST CANCER

 
BREAST CANCER

cancercancer
Breast cancer is a major health concern. An estimated 216,000 new cases of invasive breast cancer are reported each year in the U.S. resulting in more than 40,000 deaths (Brueggemeier, 2005). More than a million women throughout the world were diagnosed with breast cancer in the year 2000. The average age of diagnosis is 60. Breast cancer incidence is increasing in men as well. A better understanding of breast cancer and new treatments are improving long term survival rates. Since 1990, mortality rates due to breast cancer have dropped and, as a result, lung cancer is now the leading cause of cancer death in women (Ponzone, 2005; Darbre, 2006). In the U.S. there are an estimated 2.5 million women who have recovered from breast cancer (Batur, 2006).
The US National Cancer Institute has reported that the frequency of a number of cancers is increasing, including breast cancer, prostate cancer, testicular cancer, and brain cancer. Sixty years ago, a woman's likelihood of developing breast cancer was 1 in 22; it is now 1 in 8 (Maffini, 2006). The frequency of breast cancer has increased by 24% since 1970 (Thyagarajan, 2004). Breast cancer rates are highest in developed, industrialized regions such as the United States and Northern Europe and lowest in East Asia, Africa, and South America (Hulka, 2005). Women in industrialized nations with a Western lifestyle experience breast cancer at rates five times higher than those of developing countries (Nkondjock, 2004).
What causes breast cancer? Although a diversity of factors will be outlined in this document, the lifelong exposure to estrogen seems to be the primary risk factor of breast cancer (Darbre, 2006).

breast

1) ESTROGEN
All steroid hormones are synthesized in pathways which begin with cholesterol and many steroid hormones are synthesized from other steroid hormones. The following diagram depicts the importance of P450 enzymes in catalyzing steps involved in the production of a variety of steroid hormones (including estrogens, androgens, and progesterone) from cholesterol.

STEROID METABOLISM

(after Baker, 2004).

Estrogens are important hormones which function in both male and female bodies. Both males and females require the action of estrogen in the development of normal skeletal morphology. Estrogen affects both the testis and prostate in males. In women, estrogens promote the growth and differentiation of female reproductive structures, maintain bone mass, serve to lessen cardiovascular disease, and influence mood, cognition, and hormone release.
Estrogen is synthesized from androgens in both men and women by the gonads, brain, adipose, and the liver by the aromatase cytochrome P450 19 enzyme. CYP2C19 is also known as aromatase and estrogen sythetase. It converts androgens into estrogens and has a role in producing gender differences in adipose distribution (by being activated in some regions in women but not men). Mutations in this gene cause abnormalities in sexual differentiation and maturation and, in women, can cause amenorrhea, polycystic ovaries, and possibly virilization as well (OMIM, Osterlund, 2001).
Breast adipose and breast cancer cells can synthesize its own estrogen and thus the levels of estrogen in breast tissue can be higher than that in the plasma. Estradiol levels in the breast tissue of postmenopausal women is actually comparable to those of premenopausal women despite a 50 to 100-fold drop in plasma estradiol levels (Cavalieri, 2006; Chen, 2005).

aromatase
Aromatase is primarily produced in the ovaries of women prior to menopause, the placenta, and adipose of both men and women. Aromatase is also produced in breast tissue and is often observed to be greatest in and around tumor tissue. Aromatase has multiple promoters (the part of a gene which activates it) and cancer cells often utilize a promoter which is activated by cAMP pathways rather than the normal promoter which is primarily activated by glucocorticoids and cytokines (Brueggemeier, 2005; Miller, 2006; Sonne, 2005).

cytochromesaromatase
Aromatase inhibitors have been more successful than tamoxifen (which antagonizes estrogen action at its receptor) in the treatment of breast cancer (Goss, 2004; Miller, 2005; Howell, 2005). New aromatase inhibitors (the "third generation" of aromatase inhibitors) are being used in treatment of breast cancer. Their widespread use may significantly increase health care costs of treating breast cancer (Lonning, 2005).
Estrogens can be converted into other substances. Estradiol can be metabolized into compounds such as 4-OHE2 and 16-OHE1 which promote cell growth (but only at higher concentrations than that required by estradiol) and 2-OHE2 which does not promote cell proliferation (Seeger, 2006).

other substances
Estrogen metabolism can produce molecules such as catechol estrogen-3,4-quinones (CE-3,4-Q). Catechol estrogens are major metabolites of estrogen and, after being oxidized, they can react with DNA and lead to mutations (of adenine to guanine and guanine to adenine). Experiments with mice have shown that estrogen can cause mammary cancers even in mice which do not express estrogen receptors. The creation of catechol estrogens is accomplished through with reactions with P450 enzymes (P4501A/3A and 1B1) and further oxidation occurs through reactions with peroxidase, P450 enzymes, and glutathione S transferases. As a result, estrogen is not only relevant in cancer studies because of its effects on cell division, but also because estrogen metabolites themselves are mutagenic. Synthetic estrogens can also be converted into carcinogenic catechol metabolites. These estrogen metabolites can initiate breast, prostate, and other cancers (Cavalieri, 2006).
Because the cytochrome P450 enzymes control the synthesis and conversion of estrogens, variations (polymorphisms) in these genes and their expression levels are relevant to determining breast cancer risk. Polymorphisms in several genes which synthesize or metabolize estrogen (CYP1A1, CYP17, CYP19, CYP1B1, and COMT) have been associated with breast cancer (Dumitrescu, 2005).


ESTROGEN RECEPTORS
Nuclear receptors include receptors for estrogen, testosterone, glucocorticoids, mineralocorticoid, thyroid hormone, vitamin D and retinoic acid receptors. They all form part of a gene family derived from an ancestral protein with ligand-binding and DNA-binding domains (Amero, 1992, Zilliacus, 1994; Carosa, 1998) and a large number of orphan receptors whose function is not yet known (Detera, 1994). Most nuclear receptors have two zinc finger domains. Unlike most hormones, steroid and thyroid hormones enter cells rather than binding only to cell membrane receptors. Once they have bound their intracellular receptor, the steroid hormone-receptor complex travels to the nucleus where it binds DNA and effects gene transcription. The receptor subunits can form homodimers with themselves or heterodimers with other subunits to bind DNA molecules (OMIM).

receptormutant
Estrogen receptor (ER) genes vary in human populations. Variations in ER alpha have been linked to bone density variations in women and men and the onset of menopause in women. Human mutations suggest that ER alpha may offer some protection from heart disease and ER beta may offer some protection from prostate cancer in males (OMIM). In normal breast tissue, only about 2% of cells express ER alpha and progesterone receptors. About 70% may express ER beta but this receptor may antagonize the effects of ER alpha on cell proliferation (Kuhl, 2005). Both ER alpha and beta are expressed in the majority of breast cancers (Kuhl, 2005).

receptors
About 95% of breast cancers are hormone dependent at first. Many of these tumors gradually become hormone independent over several years, perhaps due to mutations in estrogen receptors which allow activity even in the absence of estrogen (Pasqualini, 2005). Mutant estrogen receptor proteins can differ in how they interact with estrogen and the anti-estrogens used in breast cancer therapy (Komagata, 2006). Estrogen receptor pathways can become hypersensitive to low levels of estrogen available during endocrine treatment and continue to promote cell proliferation (Johnston, 2005). Fulvestrant (Faslodex) binds to estrogen receptors, changes their shape, and promotes their breakdown rather than interaction with DNA. Unlike tamoxifen, estrogen receptor levels drop and resistant cells can not adapt to the point where the treatment actually promotes their growth (Johnston, 2005; Robertson, 2005).
Although nuclear receptors are primarily known for their ability to bind DNA and activate transcription, it is now known that they can perform other functions as well, such as direct interaction with membrane receptors. In addition to their roles in promoting gene transcription, estrogen receptors (alpha and beta) participate in pathways outside the nucleus which activate proteins such as adenylate cyclase, guanylate cyclase, nitric oxide synthase, ERk, Akt, and MAPK cascades (Pietras, 2005; Geffroy, 2005). Activated estrogen receptors can directly facilitate ion channels, IGF (insulin-like growth factor) receptors, and EGFR/HER2 (epidermal growth factor family) receptors (Johnston, 2005; Geffroy, 2005).
ADDITIONAL FACTORS WHICH CAN INCREASE LIFELONG EXPOSURE TO ESTROGEN
AGE OF MENARCHE/MENOPAUSE
Since estrogen exposure is a primary risk factor in breast cancer, the number of menstrual cycles that a woman will undergo in her lifetime is a factor in determining this risk. The lifetime number of menstrual cycles in increased by an earlier the age of menarche and a later age of menopause.
Twin studies suggest that the age of menarche is affected by genetics although nutrition, exercise, and sociological factors also play a role (Long, 2006). Variations in the receptor for LH (leutenizing hormone) can affect the onset of puberty and cause precocious puberty (OMIM).

CYP1B1 is one of the cytochrome P450 enzymes that metabolizes estrogen. Some polymorphisms of this gene are associated with later age of menopause (Long, 2006).

Tall stature is a risk factor for breast cancer and an earlier age of menarche (Waard, 2005).

ENVIRONMENTAL AND SYNTHETIC ESTROGENS
Estrogen receptors are promiscuous and can bind to a variety of compounds. As a result, a number of structurally unrelated molecules originating in sources as varied as plants and plastics can produce molecules which simulate the effects of estrogen in breast tissue (Wiseman, 2005).
Phytoestrogens are plant compounds which can either mimic the effects of estrogen in binding estrogen receptors or alter the concentration of estrogen receptors. Additional effects can include altering the concentration of steroid hormone binding globulin in the blood, acting as antioxidants, or inhibiting tyrosine kinases or cell growth (Matsuma, 2005). Environmental estrogens are thought to be factors which are increasing the rates of breast cancer, decreasing average sperm counts in men (although more comprehensive studies which take regional differences in sperm counts into consideration are needed), and contributing to other problems such as increasing the frequency of hypospadias, cryptorchidism, changes in human gender ratio at birth, and the decreasing age of pubertal onset (Safe, 2005).
Genistein and daidzein are found in legumes such as soy, 8-prenylnaringenin is found in hops, and coumestrol is found in alfalfa (Matsuma, 2005).
Cow's milk contains estrogens and the estrogen content has increased because modern dairy practices typically utilize pregnant females for milk production. Cow milk thus can increase estrogen exposure and the risk of estrogen-dependent cancers (Ganmaa, 2005).
Breast adipose can accumulate fat-soluble pesticides and chlorinated biphenyls from food, air, and water contamination. Organochlorides, atrazine, nonylphenol, and other contaminants have been shown to cause breast cancer in animals. Although a variety of these molecules have been isolated from breast milk and breast adipose tissue, it is not yet known how great a risk is posed by the measured concentrations. Breast cancer tissue can possess elevated levels of organochlorine and PCBs (Darbre, 2006).
Bisphenol-A (BPA) is a synthetic component used in the manufacture of the plastics which compose food and drink containers (including baby bottles) and medical equipment. An estimated 100 tons are released into the atmosphere per year and it can be subsequently found in drinking water. BPA can bind estrogen receptors and mimics some of the effects of estrogen. BPA has been reported in blood serum, breast milk, and the placenta. Amniotic fluid may actually have levels of BPA which are five times higher than that of maternal blood (Maffini, 2006).
Animal studies have demonstrated that BPA can lower the age of puberty, affect sexual development, promote and alter breast development, and cause abnormalities of the reproductive organs. Higher levels of BPA have been reported in women who suffered a miscarriage (Maffini, 2006).
Girls exposed to elevated levels of polybrominated byphenyls (PBBs) underwent menarche at an earlier age than normal (Maffini, 2006).
Exposure to DDT in girls has been associated with elevated levels of breast cancer later in life (Maffini, 2006).

HORMONE REPLACEMENT THERAPY
After menopause, the reduction of ovarian hormones such as estrogen can cause a variety of undesirable effects in older women such as bone loss and osteoporosis. Estrogen and progesterone affect mood and susceptibility to depression. Many women undergo depression during menopause (Rohr, 2002). Although hormone replacement therapy (HRT) can alleviate many of these problems, HRT increases the risk of breast cancer (Coombs, 2005; Hulka, 2005). In hormone replacement therapy, the risk of breast cancer was greater in women treated with a combination of progestins and estrogen compared to those treated with estrogen alone (Kuhl, 2005).
Low doses of estrogen can be applied to the vagina to reverse the atrophy caused by estrogen deprivation without significantly raising serum estrogen levels (Ponzone, 2005).
After menopause, androgen therapy has also been used to combat abdominal obesity and depression and to raise libido (Rohr, 2002). Although there are benefits to testosterone therapy in postmenopausal women, the use of testosterone alone in hormone replacement therapy is not recommended because of the potential of its conversion to estrogen in the breast (Somboonporn, 2004).

ORAL CONTRACEPTIVES
The use of oral contraceptives has either been reported to result in no risk of breast cancer or a very slight risk (Hulka, 2005). Some reports have concluded that oral contraceptive use is only likely to contribute to breast cancer risk when taken for prolonged periods or from a young age (Gompel, 2004). There may not be any increased risk when oral contraceptives are taken after the age of 20 while use prior to the age of 20 increases both the risk of breast cancer and of mutations in the BRCA1/2 genes. Oral contraceptive use prior to the age of 30 seems to increase breast cancer risk in women who have a BRCA1 mutation (but not BRCA2 mutations) (Jernstrom, 2005).
A variety of synthetic estrogens have been used as substitutes for estrogen. Ethinylestradiol (EE) is the primary estrogen agonist in oral contraceptives while estradiol and conjugated equine estrogens (CEE) are used in hormone replacement therapy (Bennik, 2004).

Diethylstilbestrol was an early non-steroidal estrogen whose use has been largely discontinued because of its association with birth defects, breast cancer, and other problems (Bennik, 2004). Women who were exposed to DES as fetuses have an increased risk of breast cancer.

ANTIPERSPIRANTS AND COSMETICS
The majority of breast cancers occur in the upper outer quadrant of the breast (31% measured according to early studies; as many as 60% in modern studies), suggesting that cosmetics or deodorant may be implicated in causing breast cancer. Some cosmetic components (such as the preservative parabens) have been isolated from breast tissue while others (such as triclosan of deodorants, phthalates, and sunscreens) have been isolated from breast milk. A number of cosmetic components (parabens, cyclosilozanes, triclosan in deodorant, sunscreens, and the athraquinones of aloe vera) display estrogen-like activity. Aluminum salts from antiperspirants affect estrogen activity. Cosmetics which increase the size of the breast (8-prenylnaringenin and miroestrol) act as estrogens (Darbre, 2006). A number of metals, including aluminum, are known to interfere with estrogen function (Darbre, 2005).
A number of benign abnormalities of the breast, such as fibroadenoma and cysts, are more common in the upper outer breast quadrant as well. Cosmetic or antiperspirant use (especially an overuse of antiperspirant) may induce abnormalities which increase the likelihood of cancer (Darbre, 2006).
Antiperspirants are often included as ingredients in other cosmetics (Darbre, 2005).

 

OBESITY
Obesity is a more serious risk factor for the development of breast cancer than other risk factors such as hormone replacement therapy, early menarche, late menopause, late birth of first child, and alcohol intake (Kuhl, 2005). Adipose cells can synthesize their own estrogen. In addition, women with central obesity tend to have lower levels of sex hormone binding globulin (SHBG) than those with peripheral obesity, perhaps because higher insulin levels in women with central obesity inhibit hepatic SHBG synthesis. This exposes tissues to higher levels of free estrogens and androgens (Pasquali, 2006).

CIGARETTES
While carcinogens from cigarette smoke have been identified in breast tissue, cigarette smoking is associated with reduced body weight and earlier menopause, both of which may reduce breast cancer risk (Hulka, 2005).

2) FACTORS OTHER THAN ESTROGEN
PROGESTERONE AND PROGESTINS
Does progesterone promote or inhibit breast cancer? Does the use of progestins in oral contraceptives and hormone replacement therapy increase or decrease cancer risk? Unfortunately, there is conflicting data on these questions. Some data may be contradictory because progesterone can be metabolized into different compounds with differing functions, synthetic progestins may differ in their function from natural progesterone, and some cancer cells may be by nature abnormal and respond differently to progesterone than normal cells.
Progesterone can be metabolized by the enzyme 5 alpha reductase to produce several 5 alpha pregnanes and the hydroxysteroid enzymes to produce 4-pregnenes. Breast cancer cells can increase the activity of 5 alpha reductase (producing more 5 alpha pregnanes than normal breast tissue) and decrease the activity of hydroxysteroid enzymes (producing less 4-pregnenes). 5 alpha pregnanes have been shown to increase breast cancer proliferation while 4-pregnenes inhibit breast cancer cells. This observation may explain why some studies of progesterone have concluded that it increases breast cancer risk while others have concluded the opposite (Wiebe, 2005; Sitruk, 2004).
While synthetic progestins have been shown in some studies to increase the risk of breast cancer (Moore, 2006), this does not seem to be true of natural progesterone. While estrogen treatment confers less risk of breast cancer than estrogen + progestins, the addition of progestins decreases the risk of cancer compared to estrogen alone (Campagnoli, 2005a). Although progestins have been shown to cause the proliferation of come cancer cells, this may have been the result of the use a synthetic molecule and the abnormal nature of the cancer cell line (Gompel, 2004).
Studies have varied in their reports of the period of the cell cycle in which cell division rates are highest in the breast (Sitruk, 2004). Some have concluded that in the luteal phase of the menstrual cycle when progesterone levels are elevated, apoptosis occurs in the breast. Apoptosis is necessary since breast cell cannot be shed as are endometrial cells (Sitruk, 2004).

ANDROGENS

androgens
Estrogen can interact with cell membrane receptors to protect cells from apoptosis. In contrast, androgens can interact with membrane receptors and can promote apoptosis (Kampa, 2005). Unfotunately, circulating androgen can also be used by breast cells to synthesize estrogen and, as a result, the relationship between androgens and breast cancer risk is not straightforward. Breast tumors whose growth is not dependent on estrogen receptors may produce androgen receptors. Although the activation of androgen receptors in normal cells inhibits cell growth, it can actually stimulate the proliferation of abnormal cells from tumors (Smith, 2006).
Testosterone can be converted into dihydrotestosterone by the enzyme 5 alpha reductase. DHT can subsequently be converted into compounds which can activate the estrogen receptor such as androstane 3alpha, 17beta-diol and androstane 3beta17beta-diol. Inhibitors which block 5 alpha reductase (such as finasteride) can limit the growth of breast cancer cells which are not affected by aromatase inhibitors (Ishikawa, 2006).

PROLACTIN

prolactin
Prolactin can increase cell division and growth in breast cancers (in part by its increase of cyclin D1 expression). Prolactin can be produced within the breast (and other tissues such as the uterus and T cells) where it acts as a paracrine hormone (Tworoger, 2006).

NEUREGULIN

NEUREGULIN
Neuregulin is a growth factor which can both cause the initiation and progression of breast cancers. Neuregulin activates the Epidermal Growth Factor/ErbB family of receptors which are classified as proto-oncogenes because of their roles in a number of cancers, including breast cancers (Amin, 2005).
Elevated expression of ErbB2 receptors is a feature of 20-30% breast cancers and is associated with decreased likelihood of survival. ErbB2 must bind to other EGF receptors which have bound their ligands in order to function. Ligands which can activate EGF family members include epidermal growth factor EGF, neuregulins 1 through 6, amphiregulin, epiregulin, epigen, TGF alpha, betacellulin, and heparin-binding EGF-like growth factor (Amin, 2005).
Neuregulin binds to ErbB3 and ErbB4 which can subsequently activate ErbB2. Its expression is observed in cancers of the breast, ovary, prostate, and thyroid (Amin, 2005).

LEPTIN AND ADIPONECTIN

hormonesfat
Some breast cancer cells express leptin receptors and leptin stimulates their growth (Dieudonne, 2002). Although elevated leptin and leptin receptor levels have been found in breast cancers, no human leptin or leptin receptor polymorphisms have been strongly associated with breast cancer (Woo, 2006).
Adiponectin is a hormone produced by adipose which reduces risk of breast and endometrial cancers. Breast cancer cell lines express adiponectin receptors and the administration of adiponectin decreases their growth (Dieudonne, 2006).

INSULIN AND INSULIN-LIKE GROWTH FACTORS

insulin
It is thought that the higher levels of insulin associated with obesity are the cause of the increased risk of breast cancer (Kuhl, 2005). In older women, increased insulin levels are associated with breast cancer (Kuhl, 2005). Hypersecretion of insulin is one of the features of type 2 diabetes. Type 2 diabetes affects about 6.5% of Americans and as many as 16% of older breast cancer patients also suffer from diabetes. Diabetes increases the risk of a number of cancers, such as breast, endometrial, colon, and pancreatic cancers (Wolf, 2006).
Insulin-like growth factors increase cell division in breast cancer cells. Adults who suffer from hypersecretion of growth hormone and insulin-like growth factors in acromegly suffer from increased rates of breast cancer and survivorship is reduced. IGF-1 functions includes its primary role as an effector signal for growth hormone (Helle, 2004).
IGF1, also known as somatomedin C, is expressed at higher levels in those with a higher risk of breast cancer before menopause (OMIM). Breast cancer rates are 7 times greater in women whose IGF-1 levels fall in the upper third of the population distribution than those in women with IGF-1 levels in the bottom third (Jenstrom, 2001). Breast cancer incidence is higher in African American women. This may be due to polymorphisms of the CYP3A4, IGF1, and AIB1 genes which are more common in African Americans. It has been observed that IGF-I (insulin-like growth factor 1) levels are higher in African American women and, unlike the situation in Caucasian women, IGF-1 levels increase with oral contraceptive use rather than decrease (Jenstrom, 2001).


EPIDERMAL GROWTH FACTOR FAMILY RECEPTORS (EGFR/HER2)

egf
The genes of the EGFR/HER gene family (EGFR, HER-2, HER-3, and HER-4) can promote the advance of cancer cells. EGFR and HER2 signaling pathways promote growth in normal breast cells and cancer cells which can proliferate even during anti-estrogen treatment (Johnston, 2005).
EGF and HER-2 activate a variety of kinases and transcription factors including MAP kinases, PI3/Akt kinases, Ras, Raf, and MEK (Pietras, 2005; Johnston, 2005).
Tumor cells exposed to anti-estrogens respond over time by increases in the expression of EGFR. As tumor cells become resistant to anti-estrogens, their invasiveness increases. EGRF/HER2/IGF-IR pathways are thought to be involved in both anti-estrogen resistance and increasing malignancy (Nicholson, 2005). Genetic polymorphisms of Her2 have been associated with increased risk of breast cancer (Kalemi, 2005).

COX AND AROMATASE
Cyclooxygenase (COX) enzymes can convert arachidonic acid to prostaglandins such as PGE2 which promote proliferation of breast cancer cells (through cAMP pathways). Thus increased COX expression can produce the local hormones which increase aromatase expression (Brueggemeier, 2005). Nonsteroidal anti-inflammatory drugs (NSAIDS such as ibuprofen) and COX inhibitors can be used to decrease estrogen production in breast tissue (Brueggemeier, 2005).

KINASES
A typical cell in the human body has the ability to respond to change. At any specific instant, a cell might be required to divide, increase the rate of its metabolic reactions, induce inflammation, stimulate cells around it, synthesize new proteins, etc. Many of the enzymes which determine these responses to a new stimulus are present in the cell prior to their activation. Therefore, one of the major aspects of gene regulation is the regulation of proteins which already have been synthesized and are present in the cytoplasm. Perhaps the most important mechanism in the post-translational regulation of protein activity is the addition or removal of phosphate groups on amino acids of these proteins. Many intracellular proteins are "off" until phosphate groups are added to it, at which point they are "on". These phosphates are added to specific amino acids of the protein (such as serine, threonine, and tyrosine).

KINASE ACTION
Reversible protein phosphorylation is essential in signal transduction (serving as the intermediate between external signals and intracellular responses to these signals). The enzymes which transfer phosphates to these amino acids, usually from ATP, are called protein kinases. Protein kinases compose 1-3% of the genes in eukaryotic genomes and are one of the largest families of enzymes. There are two major groups of protein kinases which differ in the amino acid to which phosphate groups are added: protein tyrosine kinases (which can be further divided into receptor and nonreceptor PTKs) and serine/threonine kinases. Protein tyrosine kinases are major signaling factors in animals and abnormal expression of these enzymes cause a number of human diseases (Gu, 2003).
MITOGEN ACTIVATED KINASES
MAP KINASES
MAP kinase pathways very important in the control of cell division and differentiation. They can phosphorylate diverse targets ranging from transcription factors to cytoskeletal proteins. The MAPK family has 3 subgroups: the extracellular signal kinases (ERKs), the stress-activated protein kinases (SAPKs) and the MAP3K subgroup (Kultz, 1998). EGF can activate the estrogen receptor through the MAPK pathway and estrogen receptors can activate Src which in turn activates the MAPK pathway (Geffroy, 2005). Progestins can inhibit the MAPK cascade (Gompel, 2004).
Protein kinases which promote breast cell growth include Src and focal adhesion kinase (Fak) (Planas-Silva, 2006). Estrogen receptor, IGFR, and EGFR/HER2 can stimulate breast cell growth utilizing PI3-K pathways which then activate AkT. Breast cancer treatment can involve inhibitors of AkT (Johnston, 2005; Geffroy, 2005). Other treatments such as tyrosine kinase inhibitors, farnesyltransferase inhibitors, and monoclonal antibodies can act on the signal cascade activated by EGFR/HER2 (Johnston, 2005).
Checkpoint kinases also function in breast cell proliferation. CHEK1 mutations lack the G2/M checkpoint following damage to DNA. CHEK2 is activated after DNA damage. Mutations cause the Li-Fraumeni Syndrome and may be involved in some bone marrow, breast, and prostate cancers.

One of the proteins whose overexpression is often found in breast cancer is the signal transducer and activator of transcription 3 (Stat3). Stat3 expression is induced by Src, activated epidermal growth factor receptors, JAKs, and other signals (although its expression may be constitutive in cancer cells). Phosphorylated Stat3 subsequently induces the expression of a variety of proteins including apoptosis inhibitors, angiogenesis factors (such as VEGF, COX-2, and MMP proteins), and factors which promote cell cycle progression (including Myc and Fos) (Hsieh, 2005).

TRANSCRIPTION FACTORS
At some point, cellular signaling pathways can activate new gene transcription through transcription factors. Activated estrogen, progesterone, and androgen receptors are transcription factors, as are a number of other proteins which function in breast cell division. A transcription factor complex known as AP-1 is composed of a dimer made by separate transcription factors (such as Jun, Fos, or related proteins). Many of estrogen's effects are mediated through the activation of AP-1 factors (such as the activation of cyclin D1 and c-myc). Mutations in AP-1 inhibit breast cell growth in mice (Shen, 2006).

CYCLIN
Cyclins are proteins which promote the progression of cells through various stages of the cell cycle. In the early G1 stage of the cell cycle, a restriction point is reached in which extracellular signals determine whether the cell progresses into late G1 and cell division or exits the cell cycle in G0 (Ho, 2002). In order to continue with the cell cycle, two cyclins function in the G1 phase: cyclin D which functions in the middle of G1 and E which functions at the transition between G1 and S. Cyclin D1, cyclin D2, Cdk4, and Cdk6 are protoncogenes whose misexpression can cause inappropriate progression through this checkpoint.
D cyclins interact with growth factor pathways and determine response to mitogens. D cyclins then activate cyclin E through the CIP/KIP family, particularly p21 and p27. In response to DNA damage, p53 causes the accumulation of p21 which destroys cyclin D and prevents progression through G1. The G1 checkpoint occurs through destruction of cyclin D (Agami, 2002; Ho, 2002).
Cyclin D1 mediates the increased cellular proliferation which results from estrogen receptor activity. Molecules which simulate estrogen action also increase the activity of cyclin D1 and cyclin D1 activity promotes proliferation in tumor cells which are estrogen independent but dependent on estrogen receptors (Kilker, 2004). Estrogen increases the concentration of several cyclins in breast cells and decreases the concentration of cyclin kinase inhibitors (such as p21 and p27) (Gompel, 2004). Progestin decreases the production of cyclins D1, D3, and E which control the entry into the G1 phase of the cell cycle (Gompel, 2004).

BRCA

brcabrca
Breast cancer risk can be inherited and it is estimated that less than 10% of breast cancer cases are caused by inheritance (Hulka, 2005). Women whose mothers or sisters have contracted breast cancer experience a 2-3 fold increased risk compared to women without a family history (Hulka, 2005). Some women with a family history of breast cancer choose to remove both breasts, a procedure which reduces cancer risk by about 90% (Kuhl, 2005). Although there are a number of genes which may contribute to inherited risk, the primary genes identified so far are BRCA1, BRCA2, and p53.

BRCA1 was first discovered in 1994. Mutant BRCA1 alleles are associated with increased risk of breast, cervical, uterine, prostate, and pancreatic cancers (Rosen, 2006). Mutations in the BRCA1 gene can be associated with a lifetime risk of breast cancer as high as 85% (Hulka, 2005). Those who carry a mutation for BRCA1 have a 65% chance of developing breast cancer by age 70 and a 39% chance of developing ovarian cancer (Vasen, 2005). BRCA1 and BRCA2 mutations are involved in about 20% of inherited breast cancers. The penetrance of BRCA1 mutations is estimated at 65% while that of BRCA2 mutations is estimated at 45% (Imyanitov, 2004). Heterozygotes for mutations in BRCA1 or BRCA2 also are at a higher risk for ovarian, prostate, laryngeal, digestive, liver, and pancreatic cancer (Nkondjock, 2004). BRCA polymorphisms which affect cancer risk can vary in frequency throughout different human populations. About a third of Ashkenazi Jewish women are heterozygotes for BRCA1 or BRCA2 mutations (Jernstrom, 2005).

The exact role of BRCA1 in preventing cancer is complex given that BRCA1 is known to interact, either directly or indirectly, with more than 30 proteins. Many of these interactions involve the formation of multi-protein complexes and BRCA1 may have a scaffolding role in forming these complexes (Hohenstein, 2003).
BRCA1 can regulate the transcription of genes and two regions of the BRCA1 protein (TAD and AD1) are known to activate transcription. BRCA1 inhibits estrogen receptors and promotes the activity of androgen receptors. BRCA1 increases the transcription of a number of regulatory proteins including retinoblastoma (RB). It enhances the transcriptional activity of immune proteins such as TNF alpha and interleukin 1beta. BRCA1 increases the transcription of a number of genes which inhibit cell growth. BRCA1 increases the production of antioxidants such as glutathione-S-transferases. It stimulates tumor suppressor proteins such as BRCA2 and BARD1 (Rosen, 2006).
Transcription of new genes may require unwinding the area of chromatin in which they are located and BRCA1 is a component of a SWI/SNF-like complex which remodels and unfolds chromatin. In this chromatin remodeling, it interacts with a cofactor (COBRA1) which also inhibits estrogen receptor activity. (Rosen, 2006).
BRCA1 can promote DNA repair through homologous recombination and nucleotide excision pathways (Tutt, 2002). In response to DNA damage, BRCA1 interacts with a variety of DNA repair proteins (such as mismatch repair proteins and ATM). BRCA1 forms part of the RNA polymerase II complex and may bind to damaged DNA in order to promote the binding of other repair proteins. BRCA1 increases the activity of p53 in promoting DNA repair (although not in inducing apoptosis) (Rosen, 2006).
BRCA1 can also inhibit oncogenes such as Myc-1 and IGF1R. It also reduces both the expression and activity of telomerase. (Rosen, 2006).

BRCA2 seems to have a more limited role than BRCA1 in that functions in DNA repair utilizing RAD51 (Nkondjock, 2004; Tutt, 2002). Those who carry a mutation for BRCA2 have a 45% chance of developing breast cancer by age 70 and a 11% chance of developing ovarian cancer (Vasen, 2005).

p53

p53
The tumor suppressor protein p53 is mutated in about half of human cancers. p53 is normally inactive in cells but in response to a variety of stressful stimuli, it will block progression of the cell cycle allowing for either repair of cell damage or apoptosis. Upon activation, p53 travels to the nucleus where it functions as a transcription factor promoting the expression of genes which stop the cell cycle and/or promote apoptosis. BRCA1 may be one of the proteins which functions in determining whether p53 activation leads to DNA repair or apoptosis (Hohenstein, 2003; OMIM).
p53 also performs functions which are transcription-independent. p53 is mutated in many breast cancers (between 20 and 50%) (Kalemi, 2005; Liang, 2005). Genetic polymorphisms of p53 (such as ARG72Pro and others) have been associated with increased risk of breast cancer (Kalemi, 2005; Norma, 2004).

OTHER PROTEINS OF DNA REPAIR
Other mutations in proteins which function in DNA repair are linked to breast cancer, in addition to those of BRCA and p53. Many cases of breast cancer are associated with mutations in genes involved in mismatch DNA repair, such as MSH2 and MLH1. Mutations in these genes may be present in a third of sporadic breast tumors and are associated with the degree of malignancy (Murata, 2005). ATM, p53, CHEK2, and NBS1 mutations can also cause defects in protecting cells from DNA damage and cancerous growth (Imyanitov, 2004).
The ATM (ataxia telangiectasia mutated) kinase is a major factor in responses to damage to DNA and is essential to the checkpoints at the G1-S transition, in mid S, and at the G2-M transition. ATM activates other proteins such as BRCA1, p53, and CHK2 (Tutt, 2002).

TELOMERASE
The enzyme telomerase uses RNA as a template to replicate the DNA at the ends of chromosomes (and thus functions as a reverse transcriptase). Most body cells do not express telomerase and, as a result, cells can only divide a limited number of times because the chromosomes become shorter after each cell division. Cancer cells often express telomerase and thus ensure that they can continue to divide.
The natural molecule costunolide has anti-cancer, anti-inflammatory, and anti-viral activity. Costunolide decreases the activity of the telomerase complex and the expression of reverse transcriptase (the active component of telomerase) in breast cancer cells (Choi, 2005).

VEGF
A growing cancer depends on the development of new blood vessels. VEGF is one of the most prominent factors which promotes blood vessel formation (angiogenesis) which is obviously a prerequisite for a growing tumor. Mutation of p53 proteins in breast cancer cells is associated with increased expression of VEGF. Progestins increase VEGF production, apparently through the pathway which is down-regulated by p53 (Liang, 2005).

MELATONIN


Melatonin seems to inhibit breast cancer development, proliferation, and malignancy. This action may result from decreasing the expression of estrogen, functioning as an anti-oxidant, by decreasing the activity of telomerase or by promoting certain aspects of the immune system (Cos, 2006).

SHBG
About 60% serum estradiol is bound to albumin, 38% is bound to sex-hormone binding globulin, and about 2% is dissolved in plasma (Osterlund, 2001). Sex hormone-binding globulin (SHBG) inhibits the ability of estradiol to promote the proliferation of breast cancer cells. In addition to binding estradiol, SHBG binds directly to a receptor on the surface of some breast cancer cells to inhibit proliferation (Catalano, 2005).

OTHER PROTEINS
Mammary cells express sodium/iodine symport proteins which provide iodine to the fetus that is required for the normal development and function of the thyroid. Estrogen receptors stimulate the expression of this gene and this symport gene has been reported in breast cancer cells (Alotaibi, 2006).

Genetic polymorphisms of the 5,10 methyleneterahydrofolate gene (MTHFR) have been associated with increased risk of breast cancer (Kalemi, 2005).

Decreased number of CA repeats in the first intron of the ERBB1 gene is associated with a higher incidence of breast cancer (Welmicka-Jaskiewicz, 2006).

CHROMOSOMES

chromosomes
Chromosomal abnormalities are associated with a number of cancers, including breast cancer. Specifically, changes to the structure of chromosomes 1, 3, 6, 11, 13, 16, and 17 have been linked to breast cancer, as have changes in the number of chromosomes 7, 8, 12, and 20. Variations in the number of copies of chromosome 20 (both monosomy and polysomy) have been implicated in breast cancer in a number of studies. The greater the number of extra copies of chromosome 20 in tumor tissue, the poorer the prognosis for survival. Duplications of the area of 20q13 have been identified as factors in ovarian, colon, brain, pancreatic, and other cancers (Nakopoulou, 2002).
Amplification in the copy number and increased expression of a number of genes can promote breast cancer such as MYC, HER2, BCL2, VEGF, and others (Imyanitov, 2004).

INFLAMMATION
Chronic inflammation is a factor in a number of cancers. For example, asbestos fibers can increase the likelihood of mesothelioma, inflammatory bowel disease can increase the likelihood of colon cancer, and mastitis can increase the likelihood of breast cancer. Endometriosus is an inflammatory condition which increases the frequency of breast cancer 6-fold, ovarian cancer by a factor of 5-fold, and non-Hodgkin's lymphoma by 2.5-fold. Non-steroidal anti-inflammatory drugs (NSAIDs which include aspirin and ibuprofen) decrease the risk of breast cancer (Ness, 2006).
Chronic inflammation produces reactive oxygen species (ROS), cell death through necrosis, and increased rates of cell division. Increased rates of cell division increase the importance of DNA repair mechanisms. Mutations in DNA repair proteins (such as BRCA and p53) are more likely to result in additional mutations in frequently dividing cells (Ness, 2006). Levels of reactive oxygen species (ROS) like the superoxide radical are increased in many malignant breast cells (Albright, 2005).

VIRUSES
Sexually transmitted human papillomaviruses (HPV) produce several proteins (E5, E6, and E7) which can initiate tumors and increase their growth. Well over 99.5% of cervical cancers and about 50% of head and neck squamous cell cancers are caused by papillomaviruses. Breast cancer tissue can possess papillomavirus sequences, indicating that HPV may be a risk factor in breast cancer (Kroupis, 2006).
Several reports have isolated viral sequences (homologous to mouse mammary tumor virus MMTV) in a significant percentage of breast cancers (Imyanitov, 2004).

BREAST GENERAL
There are different types of cells in the breast and their abnormalities lead to different types of cancer. Estrogen receptors (ER) are expressed in differentiated cells of the breast but not in the stem/progenitor cells. ER positive tumors form from more differentiated cells. They typically require estrogen for proliferation, can be treated with anti-estrogens, and are less life-threatening. ER negative tumors form by the proliferation of mutant stem/early progenitor cells. These tumors tend to be more malignant and more life-threatening (Kuhl, 2005).


About 1% of breast cancers are classified as metaplastic carcinomas in which glandular epithelial cells transform into either nonglandular epithelial cells or mesenchymal cells. Mesenchymal cells can subsequently produce a variety of cell types including cartilage, bone, muscle, adipose, and melanocytes (Dave, 2006). The cells of breast tumors receive physical support and hormonal stimulation from the fibroblasts in the surrounding adipose tissue. Fibroblasts surrounding breast cancers can express aromatase and produce estrogen. Tumor cells can secrete factors (such as TNF and interleukin-11) which prevent fibroblasts from differentiating into adipose cells (Amin, 2006).
Increased breast density is a risk factor for breast cancer. Alcohol intake, nulliparity, and advanced or very young age at first childbirth are factors which increase breast density. Polymorphisms of several genes (CYP1A2, COMT, and UGT1A1) are associated with breast density (Dumitrescu, 2005). Increased breast density can be caused by increased levels of proteoglycans which are associated with benign and malignant changes in the breast (Kuhl, 2005). Dense breasts are more likely to contain precancerous lesions (Waard, 2005). Benign abnormalities of the breast pose a greater risk of breast cancer, especially when they occur at a younger age (Kuhl, 2005).

BREAST DIFFERENTIATION


Prior to birth, mammary epithelium proliferates into the mammary fat pad to produce a series of ducts lined by luminal epithelia and contractile, myoepithelia. Pubertal hormones stimulate additional branching. A final step of differentiation is achieved during pregnancy and lactation in which the alveolar cells which will produce milk are formed. The end of lactation is followed by large-scale apoptosis and tissue reorganization (Dontu, 2004).
It appears that the breast has not completed its development prior to the completion of the first full-term pregnancy. The later in life that this first pregnancy occurs (or the possibility that it never occurs), the more susceptible the breast is to develop cancer. If the first pregnancy occurs after an age of 35 or so, it appears this is also a risk factor for breast cancer, perhaps because of the effects of the pregnancy growth factors on a breast which has differentiated along an alternate path (Waard, 2005).
Human placental lactogen (a hormone related to prolactin and growth hormone), prolactin, and human chorionic gonadotropin represent three hormone under investigation for stimulating breast differentiation in a way that decreases breast cancer risk.
Breast feeding is associated with decreased risk of breast cancer. This may result from a reduction in the number of menstrual cycles (and thus the amount of estrogen) or because toxic chemicals which have accumulated in the breast can be released through breast milk (Hulka, 2005).
woman with infant

In BRCA1 carriers, the breast undergoes fewer changes during pregnancy and after pregnancy the breast may be deficient in milk production (Nkondjock, 2004).


ALCOHOL
Alcohol consumption is associated with increased blood estrogen levels in males and females. Alcohol increases the conversion of androgens to estrogens which can result in degrees of feminization in male alcoholics (Purohit, 2000). Alcohol consumption increases the risk of several cancers such as cancers of the oral cavity, pharynx, liver, and esophagus (Brown, 2005). Increased alcohol consumption is a risk factor for breast cancer and alcohol consumption may cause 2.1% of cases (14 thousand women per year). In countries where alcohol use is more common (such as Italy), the frequency may be as high as 10% (Dumitrescu, 2005). In human breast cancer cells, alcohol increases cell division, aromatase expression, and estrogen receptor expression (Dumitrescu, 2005).
Breast tissue synthesizes a number of the enzymes which function in alcohol metabolism such as alcohol dehydrogenase, cytochrome P450 2E1 (CYP2E1), and xanthine oxireductase. The breakdown of ethanol produces reactive oxygen species such as superoxide ions, hydroxyl ions, and hydrogen peroxide. Hydroxyl ions and oxidized fatty acids induce DNA changes. Alcohol metabolism also forms acetaldehyde which is a carcinogen and results in chromosomal abnormalities. Reactive oxygen species affect the response of mitochondria to apoptotic signals and polymorphisms in mitochondrial superoxide dismutase have been associated with breast cancer (Dumitrescu, 2005).
Human polymorphisms which affect alcohol metabolism (such as alcohol dehydrogenase genes ADH1B, ADH1C, and ADH2). Polymorphisms in the DNA repair mechanisms which repair alcohol damage (such as XRCC1) can also contribute to the risk of a number of cancers (Dumitrescu, 2005).
Between 15% and 50% of breast cancers possess mutant p53 genes. Increased alcohol intake has been associated with increased frequency of p53 mutations (Dumitrescu, 2005).


RADIATION
Those who undergo radiological studies of their thymus or lungs (as in tuberculosis) experience increased risk of breast cancer due to radiation exposure (Hulka, 2005).

DIET
A diet rich in fruits and vegetables reduces the risk of cancer. A number of vitamins (beta carotene, retinoic acid, vitamin C, vitamin D, and vitamin E) seem to offer some degree of protection from breast cancer development. Breast cancer cells have reduced their proliferation after being treated with retinoic acid and vitamin C (Kim, 2006). Retinoids can decrease proliferation of breast cancer cells and promote apoptosis. Unfortunately, high doses of retinoids are teratogenic and have other negative side effects (Toma, 2005).
Vitamin D can inhibit tumor growth. One effect of vitamin D is that it predisposes breast cancer cells to destruction by the reactive oxygen species (such as hydrogen peroxide) released by immune cells (Weitsman, 2005).
Lignans, components of flax seed, may reduce breast cancer risk by inhibiting aromatase and thus the production of estrogen (Goss, 2004).

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