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).
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.
(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,
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,
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).
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,
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,
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).
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).
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
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,
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)
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,
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,
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).
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 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 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
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,
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
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
INSULIN AND INSULIN-LIKE GROWTH FACTORS
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
EPIDERMAL GROWTH FACTOR FAMILY RECEPTORS (EGFR/HER2)
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;
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,
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).
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 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).
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).
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,
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).
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
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).
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,
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
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).
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).
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 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).
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).
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,
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,
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).
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).
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,
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).
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
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,
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).
In BRCA1 carriers, the breast undergoes fewer changes during pregnancy
and after pregnancy the breast may be deficient in milk production (Nkondjock,
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
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).
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,
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,
Lignans, components of flax seed, may reduce breast cancer risk by inhibiting
aromatase and thus the production of estrogen (Goss, 2004).