CONCLUSION Since the identification of the BRCA1 tumor suppressor gene

Since the identification of the BRCA1 tumor suppressor gene more
than a decade ago, intense research in the field has implicated BRCA1 in
a disparate array of cellular processes. Despite the explosive knowledge
of BRCA1 in the literature, there exists a disconnect between the universal
nature of BRCA1 functions and the highly tissue-specific impact
of the BRCA1 mutations on tumorigenesis. Although BRCA1 mutation
carriers have a high risk of developing breast cancer, the genetic and
nongenetic modifiers that influence the penetrance of BRCA1 mutations
remain largely unexplored. Furthermore, the atypical clinicopathological
may be independent of LOH at the BRCA1 locus in the epithelium.
The potential tumor-promoting effect of BRCA1 loss in the stromal
Weber et al. found that, on a total-genome scale, LOH in BRCA1 mutation
expression of these angiogenesis-related genes (56, 105). Therefore,
40 McCullough, Hu, and Li
features of BRCA1-associated cancer suggests an involvement of BRCA1
in suppressing specific metastatic routes for cancer progression, although
a direct role of BRCA1 mutations in metastasis remains to be discerned.
A comprehensive understanding of these outstanding issues on BRCA1-
related cancer biology will go a long way to help develop more targeted
and effective prevention and treatment of the disease. A careful examination
of the current literature has led us to the proposal of an integrative
study of BRCA1 in the context of the unique mammary gland/tumor
microenvironment. Historically, studies of BRCA1 have been conducted
in breast epithelial/carcinoma cell lines. By looking “outside the box” of
epithelial cells and interrogating the impact of BRCA1 in both mammary
epithelial and stromal cells, we may be able to understand the etiology of
BRCA1 mutation-associated tumors in a systemic way. Given the loss of
BRCA1 expression in many sporadic breast cancer cases, continued
work in this direction also promises to have a broad application to breast
cancer therapies.
ACKNOWLEDGMENTS
NIH grants (CA118578 and CA93506). Due to limited space, we apologize
to those authors whose excellent work was not cited in this review.
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46 McCullough, Hu, and Li
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THE MOLECULAR BASIS OF THE TISSUE SPECIFICITY OF BRCA1-ASSOCIATED TUMORS

The exact molecular basis for the tissue-specificity of BRCA1-related
tumors remains elusive. Furthermore, it is unclear why somatic mutations
of BRCA1 are rare in sporadic cancer cases. The highly tissue-specific
character of BRCA1-associated tumors stands in stark contrast with the
ubiquitous nature of BRCA1 expression, as well as the generality and
multiplicity of its reported functions. As reviewed above, compelling
evidence strongly implicates BRCA1 in maintenance of genome stability.
However, it remains unclear as to why deficiency of BRCA1 function in
DNA damage response, a cellular event thought to be universally important
in all cell types and both genders, would specifically increase the
risk of breast and ovarian cancers in women. Several models have been
proposed to explain the tissue-specific nature of BRCA1-associated
tumors. For example, it has been suggested that BRCA1-deficient breast
and ovarian epithelial cells may be more refractory to apoptosis than
those in other tissues, thus allowing the former to accumulate additional
genetic instability (65). Alternatively, the tissue-specific nature of BRCA1-
associated tumors may arise from a higher frequency of LOH in the
breast and ovarian epithelial cells (66). While maintenance of genetic
stability is obviously an important part of the tumor suppressor function
of BRCA1, it remains to be seen whether loss of this activity alone could
fully account for the tissue- and gender-specific nature of BRCA1-
associated tumors.
The action of estrogen is critical to both normal mammary gland
development and breast cancer (67–69). Aberrant changes of the expression
and/or activity of ERα and its coregulators have been associated
with breast carcinogenesis (70, 71). In light of the fact that cancerpredisposing
mutations of BRCA1 predominantly affect the breast and
3. BRCA1 in initiation, invasion, and metastasis 35
3.1 Possible tissue-specific genetic instability
3.2 Modulation of ERα activity by BRCA1instability
specificity” could be explained by a potential link between BRCA1 and
estrogen action. In support of this notion, the wild-type BRCA1 protein
has been implicated in the regulation of ERα-mediated gene expression.
Initial studies by Rosen et al. demonstrated that the exogenous
expression of BRCA1 resulted in downregulation of estrogen-stimulated
expression of an estrogen-responsive reporter construct in human breast,
prostate, and cervical carcinoma cell lines (72). Additional studies by
this and other groups have shown that BRCA1 is physically associated
with ERα-regulated promoters such as pS2 and regulates expression of
the corresponding endogenous gene expression in breast cancer cell lines
(40, 55, 73). Additional in vitro characterization has indicated that
BRCA1 and ERα physically interact with each other through the aminomay
promote estrogen-dependent cell growth and neoplasia in the breast
tissue. However, the tissue culture-based findings would have to be
reconciled with the clinical observation that most BRCA1-associated
breast tumors are basal-like and ERα-negative (see below).
In addition to dysregulated transcriptional activity of ERα, prolonged
estrogen exposure is also a well-documented risk factor for breast cancer
(68, 74–78). Ovaries, specifically ovarian granulosa cells, are the primary
source of estrogen in premenopausal women. This explains why early
menarche and late menopause are associated with increased risks of
breast cancer (79). Aromatase (Cyp19) is expressed in a restricted number
of steroidogenic tissues including ovaries. The enzyme catalyzes the conversion
from androgen to estrogen, the rate-limiting step in estrogen biosynthesis
(80). Recently published work from our laboratories suggests
that expression of BRCA1 in ovarian granulosa cells is inversely correlated
with that of aromatase during steroidogenesis (81). Importantly, small
interfering RNA (siRNA)-mediated knockdown of BRCA1 or its partner
BARD1 resulted in elevated aromatase expression and its enzymatic
activity in ovarian granulosa cells (81). In an independent study, Dubeau
et al. made an intriguing observation that ovarian granulosa cell-specific
Brca1 knockout mice develop ovarian and uterine tumors that still contain
ovary, two major estrogen-responsive tissues, the conundrum of “tissueterminal
region of BRCA1 and the ligand-binding domain (LBD) of
ER-α in an estrogen-independent manner (40). Therefore, loss of the transcriptional
corepressor function of BRCA1 in BRCA1-deficient cells
the wild-type Brca1 gene (82). These in vitro and in vivo findings point
3.3 BRCA1 and regulation of estrogen biosynthesis
36 McCullough, Hu, and Li
to a cell nonautonomous role of BRCA1 in modulating the endocrine
and/or paracrine actions of estrogen.
Figure 2. Proposed impact of BRCA1 on different cell types within the mammary tumor
At menopause, ovarian estrogen production ceases and extragonadal
sites such as adipose tissue become the prominent sources of estrogen
(80, 83). In addition to the alteration in the source of estrogen, the
capacity of estrogen as a signaling molecule changes from an endocrine
to a localized paracrine/autocrine role (84). Indeed, elevated intratumoral
aromatase expression and estrogen production are linked to the development
of postmenopausal breast cancer (85, 86). This involves an intricate
paracrine loop between tumor and the surrounding adipose stromal cells
(ASCs): tumor cell-derived factors such as interleukin 6 (IL-6) and prostaglandin
E2 (PGE2) stimulate aromatase expression and hence estrogen
production in ASCs, which in turn promote estrogen-dependent growth
of tumor cells (87-89). Such a “vicious cycle” is thought to facilitate
breast cancer progression in the unique mammary tissue microenvironment.
This also serves as the rationale for using aromatase inhibitors,
such as letrozole, as efficacious agents for the treatment of postmenopausal
breast cancer (90). In addition to the modulation of aromatase
expression in ovarian granulosa cells (81), BRCA1 also appears to
repress aromatase gene expression in ASCs (91, 92). Therefore, by
microenvironment. E2 and T stand for 17beta estradiol and testosterone, respectively.
blunting estrogen production in ovaries and mammary microenvironment,
BRCA1 may reduce estrogen-mediated gene expression and
3. BRCA1 in initiation, invasion, and metastasis 37
suppress the initiation of estrogen-dependent tumorigenesis (Fig. 2). This
function of BRCA1 in stromal cells may occur in parallel with the
BRCA1-mediated repression of ERα transcriptional activity in mammary
epithelial cells. Given the known carcinogenic effect of estrogen
and its metabolites (93), elevated local estrogen levels due to BRCA1
deficiency in stromal cells may also contribute to genetic instability, thus
compounding the consequence of impaired DNA repair capability in
BRCA1-defective epithelial cells within the same microenvironment.
4.
The relevance of estrogen/ERα to the etiology of BRCA1-associated
tumors has been a long-standing clinical conundrum. BRCA1-associated
tumors are largely ERα-negative (6) and their gene expression profile
resembles that from basal epithelial cells in the mammary gland (94, 95).
On the other hand, prophylactic oophorectomy, which removes the major
source of circulating estrogen in premenopausal women, significantly
reduces risk of breast cancer in BRCA1-mutation carriers (96, 97).
Consistent with the findings in human (96, 97), oophorectomy decreases
the incidence of mammary tumor formation in the MMTV-BRCA1-/-
mouse model (98). In addition, tamoxifen has been shown to be effective
in reducing the risk of contralateral tumors in BRCA1-mutation carriers
(99). Epidemiological evidence also suggests that hormonal exposure
and obesity in adolescence, which are well-known risk factors for
sporadic breast cancer, can significantly affect breast cancer onset for
BRCA1-mutation carriers (12).
How could one reconcile the ERα-negative feature of BRCA1-
associated tumors with the apparent impact of estrogen exposure on the
disease risk? One possible explanation for the aforementioned paradox is
that ERα-positive BRCA1-deficient cells may evolve to become ERα-
negative tumors during the disease progression. Consistent with this notion,
OF BRCA1-RELATED BREAST CANCER
4.1
early-stage mammary tumors from MMTV-BRCA1-/- knockout mice are
largely ERα-positive, whereas late-stage tumors usually lack ERα expression
(100) (Chuxia Deng, NIH, personal communication). Therefore, it is
MOLECULAR BASIS FOR
CLINICOPATHOLOGICAL FEATURES
Is BRCA1-associated tumorigenesis
estrogen-dependent?
38 McCullough, Hu, and Li
possible that modulation of estrogen production and/or the transcripttional
activity of ERα by wild-type BRCA1 in stromal and epithelial
positive cells in the same microenvironment could influence the behavior
of BRCA1-deficient, ERα-negative preneoplastic cells through a paracrine
mechanism. Obviously, an in-depth investigation of the BRCA1–
BRCA1-associated tumors are usually diagnosed as high-grade
infiltrating ductal carcinoma (99). Patients with BRCA1-associated breast
tumors tend to have a poorer prognosis than those with sporadic tumors,
suggesting that loss of BRCA1 function may lead to a more aggressive
progression of breast cancer. Interestingly, a recent report suggests a
high incidence of brain metastasis in BRCA1-associated cancer cases
(101). Contrary to what has been observed in sporadic breast cancer,
BRCA1 mutation-associated poor prognosis often occurs in nodenegative
cases, where tumors do not spread to axillary lymph nodes (6).
It was postulated that BRCA1-associated tumors might choose metastatic
routes other than the lymphatic system, perhaps through newly formed
blood vessels surrounding the tumors (6). Just as proposed for the
initiation of BRCA1-associated breast tumors, the exact pattern and route
immortalized mammary epithelial cell line MCF10A disrupts normal
acinar morphogenesis in vitro (104). Reduction of BRCA1 in the MCF10A
cell line led to aberrant cell proliferation and failure to respond to extracellular
matrix (ECM)-dependent differentiation signals. Of particular
interest is the observation in this study that treatment of BRCA1-depleted
MCF10A cells with conditioned medium from control counterparts
of BRCA1-associated tumors. In an alternative scenario, normal ERα-
cells, respectively, may play a critical role in suppressing the initiation
estrogen connection will be of great importance to more targeted prevention
and treatment of BRCA1-associated cancers. The same research may also
shed light on the functional consequences of reduced BRCA1 expression
associated with many sporadic breast cancers (6).
for the progression and spreading of these tumors may also be determined
by an intricate interaction between BRCA1-deficient tumor cells
and the surrounding stroma. Is there any evidence in support of such
a hypothesis?
partially restored the ability of these BRCA1-depleted cells to complete
three-dimensional acinar morphogenesis in vitro. These results are
consistent with the possibility that mammary epithelial cells secrete an
4.2 Why do BRCA1-associated cancers have a poor
prognosis?
Using a recently popularized three-dimensional cell culture system
that mimics the in vivo mammary microenvironment (102, 103), Furuta
et al. showed that BRCA1 depletion by shRNA interference in the
3. BRCA1 in initiation, invasion, and metastasis 39
autocrine/paracrine factor in a BRCA1-dependent fashion to promote
normal differentiation. In support of this notion, a follow-up study from
the same group found that BRCA1 directly represses transcription of
angiopoietin (ANG1), the product of which acts in a paracrine manner
to promote endothelial cell survival and vascularization (56). In an
independent study, BRCA1 was shown to repress ERα-dependent transcription
and secretion of vascular endothelial growth factor (VGEF) in
breast cancer cells (105). Of clinical importance, both studies demonstrated
that cancer-predisposing mutants of BRCA1 fail to reduce the
these studies raise a distinct possibility that loss of BRCA1 in mammary
epithelial cells may have a significant impact on the behavior of the
stromal cells in the tumor microenvironment, which in turn may influence
the metastatic outcome of the BRCA1-associated cancer (Fig. 2).
Cytogenetic analyses of clinical samples also shed some intriguing
light on the genetic instability of BRCA1-associated tumor and the
surrounding stroma in the same microenvironment. In a recent report,
carriers was similar between the breast tumor cells and the associated
stroma (106). Further, LOH at the BRCA1 locus of several patients was
only observed in the breast tumor stroma (106). These observations
suggest a role for stromal BRCA1 in suppressing tumor progression that
compartment may be similar to that of stromal p53 mutations recently
demonstrated in breast and prostate tumors (107, 108). Lastly, it has been
recently reported that malignant human breast cancer epithelial cells can
fuse with and transform mouse stroma (109). Therefore, it will be of
interest to see whether the increased genetic instability due to loss of
BRCA1 in the microenvironment may result in fusion of the epithelial
and stroma components.

A PERSPECTIVE FROM THE TUMOR MICROENVIRONMENT

Shaun D. McCullough, Yanfen Hu, and Rong Li
Department of Biochemistry and Molecular Genetics, Health Science Center, University
of Virginia, Charlottesville, VA 22908, USA
Abstract: Women who inherit cancer-predisposing mutations in the BRCA1 gene
have about 80% lifetime chance of developing breast cancer. BRCA1
mutation-associated tumors are often diagnosed as high-grade, typically
display a basal epithelial phenotype, and proliferate rapidly. While somatic
mutations of BRCA1 are rarely found in sporadic breast cancer cases, 30–
40% of the sporadic cases show reduced BRCA1 expression, supporting
the notion that impaired BRCA1 function may contribute to the development
of both familial and sporadic forms of breast cancer. Furthermore,
low levels of BRCA1 expression have been linked with the occurrence of
distant metastases in sporadic disease. Since cloning of the gene more than
a decade ago, BRCA1 has been implicated in a large array of cellular
functions, most notably DNA damage repair. However, the relationship
between the known molecular functions of BRCA1 and the clinicopathological
features of BRCA1-associated tumors remains elusive. Why
do BRCA1 mutations predominantly affect female breast and ovaries?
Why do BRCA1-associated cancers tend to have a poor prognosis? How
can the knowledge of BRCA1 function be translated into more targeted
and efficacious therapies? In this review, we will discuss these important
issues in light of some recent findings from laboratory and preclinical
studies, which point to a need to look “outside the box” of epithelial cells
by elucidating BRCA1 functions in the context of the unique tumor
microenvironment.
Keywords:
receptor, tumor microenvironment.
AND :
BRCA1 IN INITIATION, INVASION,
BRCA1, DNA repair, transcription, estrogen, tissue-specificity, estrogen
1. BRCA1: A TISSUE-SPECIFIC TUMOR
SUPPRESSOR GENE
Breast cancer susceptibility gene BRCA1 was identified in 1994 through
genetic linkage analysis and positional cloning (1, 2). Germ-line mutations
of BRCA1 occur at a frequency of approximately 1 in 250 women,
and these mutations account for 45% of the familial breast cancer and
80–90% of the hereditary cases where both breast and ovarian cancers
occur (breast-ovarian cancer syndrome) (3–5). Genetic analysis of BRCA1-
associated tumor specimens strongly indicate that BRCA1 functions as a
tumor suppressor, as the tumors invariably lose the wild-type copy of
BRCA1 and retain the inherited mutant copy (loss of heterozygosity;
LOH). However, in contrast to mutations of other well-defined tumor
less, reduced expression of BRCA1 mRNA, and protein has been observed
in a significant percentage (30–40%) of sporadic breast/ovarian cancer
cases; and this is particularly true in tumors with high nuclear grade
(6–8). Furthermore, promoter hypermethylation-mediated gene silencing
of the BRCA1 locus occurs in 10–15% of sporadic breast and ovarian
cancer cases (9–11), supporting the notion that BRCA1 may also play a
role in suppression of sporadic breast cancer. In a recent comprehensive
analysis of cancer risks among BRCA1 mutation-carriers, it was shown
that this group of women has 80% chance of developing breast cancer in
their lifetime (12). Interestingly, the same study also found that physical
exercise and lack of obesity in adolescence significantly delay the onset
of BRCA1-associated breast cancer, which underscores the importance of
nongenetic factors in cancer prevention.
Figure 1. Diagram of the BRCA1 protein. The structural motifs including the RING and
BRCT domains are highlighted. Also listed is a subset of BRCA1-interacting proteins.
region are rarely found in sporadic breast or ovarian cancers. Neverthesuppressor
genes such as p53, somatic mutations in the BRCA1 coding
32 McCullough, Hu, and Li
2. STRUCTURAL AND FUNCTIONAL
FEATURES OF THE BRCA1 PROTEIN
The human BRCA1 gene encodes a 1863-amino acid protein, which
contains a highly conserved RING finger domain at the amino terminus
and two BRCT repeats at the carboxyl terminus (Fig. 1). The vast majority
of cancer-predisposing mutations of BRCA1 give rise to truncated and
presumably nonfunctional proteins (3). Approximately 10% of mutations
result in change of a single amino acid, many of which are located in the
RING and BRCT domains. The molecular functions of the BRCA1 protein
have been a subject of intense research for more than a decade. The
ubiquitously expressed protein is implicated in a large array of cellular
events, including DNA repair, transcription, chromatin remodeling,
ubiquitination, DNA damage checkpoint, mitotic spindle checkpoint, and
control of centrosome duplication (7, 13–21).
Among all the reported functions of BRCA1, its role in the DNA
damage response has been most extensively investigated (13, 14, 16, 18).
A wealth of evidence indicates that BRCA1 is physically associated with
multiple proteins involved in DNA repair and checkpoint control, and
their nuclear co-localization is one of the hallmarks in the activation of
DNA damage response (22–26). BRCA1 is phosphorylated by several
key protein kinases involved in the DNA damage checkpoint control,
including ATM, ATR, and CHK2 (27–29), and is thought to act as a
signal-transducing molecule that links upstream sensors of DNA damage
with the downstream effectors. BRCA1-deficient human and murine cells
are hypersensitive to various types of genotoxic insults, including DNA
double-strand breaks (30–34). Chromosomal instability due to compromised
functions of BRCA1 in DNA repair and DNA damage
checkpoint most likely contribute in a significant manner to BRCA1
mutation-associated cancer susceptibility.
In addition to DNA repair, the role of BRCA1 in gene regulation
has also been well explored (7, 13, 15, 21). Although BRCA1 is not a
sequence-specific DNA binding protein, it can be associated with a
number of site-specific transcription factors (35–41), chromatin-modifying
protein complexes (42–45), and the RNA polymerase II (RNAPII) holoenzyme
itself (42, 46–48). Ectopic expression and siRNA knockdown
experiments have led to the identification of a number of BRCA1 target
genes including p21CIP, GADD45, pS2/TFF1, MAD2, OPN, and ANG1
(35, 39, 40, 49–56). Many of the BRCA1-regulated genes are important
players in cell cycle regulation, mitotic checkpoint, cell migration, and
3. BRCA1 in initiation, invasion, and metastasis 33
angiogenesis, and their aberrant expression due to the loss of BRCA1
activity in transcription may lead to the BRCA1 mutation-associated
tumorigenesis.
So far the only known enzymatic activity of BRCA1 is its ubiquitin
(Ub) E3 ligase activity. The N-terminal RING domain of BRCA1 interacts
with another structurally similar RING finger protein BARD1, and
the RING domain of BRCA1 abolish the Ub E3 ligase activity of the
BRCA1/BARD1 complex, providing a compelling link between ubiquitythe
BRCA1/BARD1 complex remain to be elucidated. However, recent
studies have indicated that ubiquitination of the largest subunit of RNA
polymerase II by BRCA1/BARD1 is responsible for DNA damage-induced
inhibition of RNA processing (58, 59). In addition, BRCA1/BARD1 has
been shown to ubiquitinate γ-tubulin, which is involved in the control of
proper centrosome duplication and chromosomal segregation (60).
The construction of whole-body and tissue-specific BRCA1 knockout
mice has allowed for a better understanding of the role that Brca1 plays
in both embryonic development and tumorigenesis in vivo. Whole-body
BRCA1 knockout mice fail to develop properly and die in utero before
day 7.5 of gestation (61). Characterization of the embryonic lethal phenotype
in the BRCA1 null embryos suggested that they exhibited defects in
cellular proliferation (61). Further studies with this knockout mouse
model indicated that loss of functional p53 delayed embryonic lethality
participate in a common genetic pathway (62). Relatively recent work
affected both cell growth and metastatic potential in MEFs isolated from
the knockout mice (63). In this system, loss of BRCA1 results in p53-
dependent senescence, therefore allowing clonal selection for cells that
can bypass senescence through loss of functional p53. Interestingly, the
+/+
immortalized clone was shown to be p53-negative. Once immortalized,
the BRCA1 null MEFs proliferated at a significantly greater rate and
exhibited greater metastatic potential than immortalized control MEFs.
The results from these studies begin to reconcile the seeming paradox
between the accepted function of BRCA1 as a tumor suppressor and the
with the findings from the laboratory research, studies of human clinical
nation and breast cancer. The exact in vivo ubiquitination substrates of
in vitro (19, 57). Importantly, missense cancer-predisposing mutations in
the BRCA1/BARD1 heterodimer confers strong Ub E3 ligase activity
by Cao et al. demonstrated how the interplay between BRCA1 and p53
a much lower frequency than BRCA1 controls and nearly every
slow growth phenotype of BRCA1 mutant/null cells in culture. Consistent
immortalization of BRCA1-null MEFs was observed to occur with
samples indicate that BRCA1 mutation-associated breast cancers exhibit
34 McCullough, Hu, and Li
in BRCA1 null mice to day 9.5 of gestation, suggesting that BRCA1 and p53
inactivating mutations in the p53 gene with a greater frequency than their
sporadic counterparts (64).

CONCLUSIONS Metastasis, the spread of cancer

CONCLUSIONS Metastasis, the spread of cancer cells from the primary tumor to
distant organs and their treatment-resistant proliferation in multiple
locations, remains a major clinical and biological challenge.
The genetics of breast cancer metastasis is a very broad and complex
field of study. It is relatively new and expanding. There are several
metastases (90). Another study conducted by Nakopoulou et al. using
studies for relevance, and their mechanisms of action need to be
elucidated. Interestingly there is not a unique signaling pathway that has
emerged as a key. This further emphasizes the need for more exhaustive
studies. A better understanding of the molecular mechanisms that regulate
the process of metastasis and of the complex interactions between the
metastatic cells and host factors can provide a biological foundation for
the design of more effective therapy.
potential candidates identified; however, functional validation, patient
2. Genetic control of breast cancer metastasis 21
ACKNOWLEDGMENT
We wish to acknowledge all our colleagues and collaborators whose
R.S.S. is a recipient of Susan G. Komen Breast Cancer Foundation
research grant # BTCR0503488.
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with an aggressive histologic variant–invasive micropapillary carcinoma.
30 Samant, Fodstad, and Shevde

Molecules with mechanism

Molecules with mechanism yet to be confirmed COM1
human breast carcinoma cells upon formation of experimental metastatic
tumors. Using primary carcinomas and uninvolved adjacent breast tissue
mRNA were significantly upregulated in the tumors compared to the
normal breast tissues (90, 91). Jiang et al. compared a cohort of breast
cancer tumors (n-120) with matched normal nonneoplastic mammary
tion, leading to enhanced anchorage-independent growth in vitro and
COM1 was identified as a novel factor which was upregulated in
from breast cancer patients Ree et al. found that the levels of com1
3.1.8. Gene regulation (Chromatin remodeling)
Metastasis-associated genes (MTAs)
Metastasis-associated genes (MTAs) represent a rapidly growing
novel gene family. At present, there are three different known genes
(MTA1, MTA2, and MTA3) and six reported isoforms (MTA1, MTA1s,
MTA1-ZG29p, MTA2, MTA3, MTA3L). MTA1, MTA2, and MTA3 are
components of the nucleosome remodeling and deacetylation complex,
which is associated with adenosine triphosphate-dependent chromatin
remodeling and transcriptional regulation. MTA proteins, as a part of the
NuRD complex (nuclear remodeling and deacetylation complex), are
thought to modulate transcription by influencing the status of chromatin
remodeling (81–84). MTA1mRNA expression directly correlates with
metastatic potential (85, 86); however, the function of the MTA1 gene
product in tumor progression and metastasis remains unknown. Altered
expression of MTA1 has been observed in both premalignant lesion and
malignant breast carcinoma, but an elevated nuclear expression was
observed in ER-negative carcinomas. MTA3 exclusively expressed in a
subset of cells of ER-positive premalignant lesions but not in carcinomas
(87). MTA2 expression seems to be unrelated to ER status. Loss of
MTA3 expression and more nuclear localization of MTA1 occurred with
2. Genetic control of breast cancer metastasis 15
PCR. They have reported that COM1 is a nuclear protein, whose
expression is reduced in human breast cancer tissues and cancer cell
tumors correlate with the prognosis of the patients and with the longterm
overall survival in association with ER status (92, 93). Thus there
is an apparent controversy regarding COM1. The mechanism by which
COM1 acts is still debatable. However, Bratland et al. compared the
growth-regulatory mechanisms of nontumorigenic and estrogen-dependent
MCF7 cells with those of the tumorigenic and tamoxifen-resistant
subline MCF7/LCC2 in the presence of Vitamin D3. Proliferation of
MCF7/LCC2 cells, which revealed constitutive COM1 expression, was
tissues (n = 32) for COM1 using conventional and real-time quantitative
lines. The loss of COM1 protein is primarily from the nuclear compartment
in cancer cells. The expression levels of COM1 in breast
inhibited by Vitamin D3. Furthermore, when the com1-negative MCF7
cells were stably transfected with COM1, the resulting MCF7/COM1
cells showed a significant decrease in colony formation (94). These
3.2.
The MSG field was launched by the discovery of nm23 (95, 96). This
field realized its momentum at the turn of the last millennium. To date
results indicate that rather than promoting growth, COM1 may participate
in the regulatory pathway involved in cellular growth inhibition
when recruited by inhibitory signals (94).
Metastasis Suppressor Genes (MSGs)
there are at least 13 metastasis suppressor genes functionally characterized:
Nm23, KAI-1, KISS-1, TXNIP (VDUP1), CRSP3, MKK4, Src-suppressed
C kinase substrate (SSeCKS) the likely rodent ortholog of human
Gravin/AKAP12, RhoGDI2, E-cadherin (encoded by CAD1), Drg-1 (a.k.a.
RTP, cap43, and rit42), Tissue inhibitors of metalloproteases (TIMPs),
RKIP, and BRMS1; however, not all of them have been characterized for
involvement in suppression of breast cancer metastasis (97).
We must mention that most of these studies are based on using
human breast cancer xenografts in athymic mice. There are two ways of
verifying the functional impact of the metastasis suppressor genes in
cardiac injection (98, 99).
3.2.1. Breast Cancer Metastasis 1 (BRMS1)
BRMS1 has been shown to suppress metastasis of a variety of metastatic
human breast cancer lines (100, 101). The murine ortholog of BRMS1
(cells injected via tail vein and pulmonary metastasis scored). There are
some new models of breast cancer cells metastasizing to bone via intraorthotopic
mammary fat pad site) or experimental metastasis model
animal models; the spontaneous metastasis model (xenograft at the
16 Samant, Fodstad, and Shevde
a transcription co-repressor complex. There is a group of homologous
was shown to have similar properties (102). BRMS1 is a member of
involved in chromatin modulation (103). BRMS1 is implicated in regulatexpression
by targeting nuclear factor-kappaB activity (104). DeWald
and others have implicated BRMS1 reduction of phosphoinositide
signaling in MDA-MB-435 breast carcinoma cells (105). However till
date there is no convincing patient study that substantiates the exact role
of this protein or loss of expression of BRMS1.
3.2.2. KiSS1
carcinoma MDA-MB-435 cells after transfection with the MSG KiSS-1,
implicating its importance in breast cancer (106). Expression of KiSS-1
in breast cancer cells is regulated by direct interaction of transcription
hormone-related protein regulates KiSS-1 in breast cancer cells (108).
Studies by Stark and colleagues revealed significantly reduced mRNA
expression of MSG KISS-1, KAI1, BRMS1, and MKK4 in breast cancer
ing gap junctions and Cicek et al. have shown that BRMS1 inhibits gene
Lee et al. demonstrated suppression of metastasis in human breast
However, there are conflicting reports about the role of KiSS-1 in
breast cancer. When Martin et al. determined the expression and distribution
of KiSS-1 and its receptor in human breast cancer tissues to
identify a possible link between expression levels and patient prognosis,
contrary to the intuitive extrapolation from the observations of the initial
mouse model studies stated above, levels of expression of KiSS-1
were higher in tumor compared to background tissues and significantly
factors AP-2alpha and SP1 (107). Dittmer et al., showed that parathyroid
brain metastasis (109).
increased in node positive tumors compared to node negative. KiSS-1
expression was also increased with increasing grade and TNM status.
There were no such trends with the KiSS-1 receptor. Expression of
KiSS-1 was higher in patients who had died from breast cancer than
those who had remained healthy whereas expression of the receptor was
more aggressive phenotype (110). This work suggests that KiSS-1 plays
a role beyond the initial metastasis repressor in this cancer type.
NM23 is known to be a family of eight proteins occurring in all
cellular compartments (110). In vitro correlates of suppression include
reduced invasion, motility, and soft agar colonization, and induction of
differentiation. Differentiation remains one of the key correlates of altered
NM23 expression in multiple model systems. Both in vitro and in vivo
studies support a role for this gene in breast differentiation. NM23-H1
transfectants of the human MDA-MB-435 breast carcinoma cell line
formed acinar structures, secreted the basement membrane proteins
reduced. Thus, overexpression of KiSS-1 in breast cancer cells results in
3.2.3. NM23
2. Genetic control of breast cancer metastasis 17
proteins that are BRMS1-like proteins and are collectively or independently
laminin and type IV collagen to the basal side of the acinus, and
produced sialomucin in three-dimensional cultures in the laboratory of
Bissell (110). A knockout mouse for NM23-M1 exhibited growth retardation
and pronounced mammary defects. In virgin mice, ductal elongation
and branching was poor and the mammary gland failed to fill the fat pad.
These morphological differences were overcome in pregnancy, but a
functional defect persisted in feeding pups (111). The breast cancer data
support the conclusion that altered NM23 expression levels may be of
functional significance in humans.
CD82, also known as KAI1, was identified as a prostate cancer MSG
on human chromosome 11p1.2. The product of CD82 is KAI1, a 40- to
75-kDa tetraspanin cell-surface protein also known as the leukocyte cellsurface
marker, CD82. Phillips et al., demonstrated a correlation between
reduction of metastasis in the MDA-MB-435 model system and increased
expression of the Kai-1 protein (111). Downregulation of KAI1 has been
found to be clinically associated with metastatic progression in a variety
of cancers. Stark et al. revealed significantly reduced mRNA expression
of KAI1, in breast cancer brain metastasis (109). Yang et al., showed that
3.2.4. KAI1
KAI1 protein levels were inversely correlated with the metastatic potential
of breast cancer cells. Furthermore, examination of KAI1 protein expression
in specimens from 81 patients with breast cancer showed high levels
of KAI1 protein in normal breast tissues and noninvasive breast cancer
demonstrated significantly lower KAI1 expression (112).
(ductal carcinoma in situ). In contrast, KAI1 expression was reduced
in most of the infiltrating breast tumors. More malignant tumors
3.2.5. MKK4
MKK4, located in close proximity to p53 gene, is thought to be a
tumor and a MSG. A low-rate MKK4 gene alteration has been found in a
few tumor types, including breast and pancreatic cancers (113). Also, the
expression of MKK4 is significantly reduced in breast cancer brain
metastases (109). A suppressor activity for prostate and ovarian tumor
metastasis has also been suggested (114) (115). However, ectopic
expression of MKK4 by adenoviral delivery in MKK4-negative cancer
lines stimulated the cell proliferation and invasion, whereas knockdown
of MKK4 expression by small interference RNA in an MKK4-positive
breast cancer cell line, MDA-MB-231, resulted in decreased anchorageindependent
growth, suppressed tumor growth in mouse xenograft
model, and increased cell susceptibility to apoptosis brought by stress
signals such as serum deprivation (109). These results argue that MKK4
functions as a pro-oncogenic molecule instead of a suppressor in breast
tumors.
18 Samant, Fodstad, and Shevde
3.2.6. TXNIP
expressed in the breast cancer cell line MCF7, is localized predominantly
in the nucleus and exhibits growth suppressive activity. TBP-2 protein
localizes to the nucleus in cells treated with an anticancer drug,
suberoylanilide hydroxamic acid (116). Estrogen represses TXNIP in
MCF7 human breast cancer cells. This repression can be blocked by
treatment with the histone deacetylase inhibitor, trichostatin A (117). A
high-fat n-6 diet caused a decrease in the expression of VDUP1 and was
associated with a higher number of adenocarcinomas and aggressive
tumor phenotype in experimental breast cancer (in rats) (118).
3.2.7. E-cadherin
E-cadherin is the prototype member of the cadherin family of calciumdependent
cell–cell adhesion molecules. It is expressed in normal adults
in luminal epithelial cells, and is lost concomitantly with tumor protein
1 (VDUP1) is an endogenous molecule interacting with thioredoxin
(TRX), negatively regulating TRX function and being implicated in
the suppression of tumor development and metastasis. TBP-2 ectopically
connection with diet was identified by Escrich et al. who reported that a
gression in breast cancers. In fact, E-cadherin expression is irreversibly
lost in >85% of invasive lobular breast cancers. Loss of E-cadherin
appears to be an early event in these tumors, since even noninvasive
lobular carcinoma in situ frequently lacks E-cadherin (119, 120). This
may result from loss of heterozygosity (LOH) at 16q22.1, involving the
E-cadherin gene CDH1 (approximately 50%) (121), frequently in
combination with mutation (50%) (119, 120, 122) or epigenetic silencing
of the remaining CDH1 allele (123–128). The status of the estrogen
receptor (ER) can also have regulatory effects on E-cadherin. Absence of
the ER results in decreased levels of a metastasis-associated protein,
Thioredoxin-binding protein-2 (TBP-2)/vitamin D3 upregulated pro-
MTA3, which plays a role in chromatin remodeling as part of a larger
repressive complex, Mi-2/NuRD. This complex normally represses the
transcription factor Snail, which in turn represses E-cadherin. Loss of
estrogen signaling reverses the repression of Snail, resulting in its
increase and subsequent repression of E-cadherin (129–133). Loss of
E-cadherin correlates with ER negativity, supporting this as one possible
mechanism for E-cadherin loss in some breast cancers. In general, while
E-cadherin expression correlates inversely with histological grade and
thus differentiation, its expression is not well correlated with survival. In
some studies reduced E-cadherin correlates with shorter metastasis-free
other reports indicate that heterogenous staining of the tumor for
E-cadherin is a poor indicator. In contrast, other studies suggested that
E-cadherin presence was actually a marker of poor survival. In fact, cells
periods and poor prognosis in node negative patients (124, 134), while
2. Genetic control of breast cancer metastasis 19
3.2.8. Drg-1
The expression of the Drg-1 (differentiation-related gene-1) protein is
significantly reduced in breast tumor cells, particularly in patients with
lymph node or bone metastasis as compared to those with localized
breast cancer. In studies by Bandopadhyay et al. Drg-1 expression also
exhibited significant inverse correlation with the disease-free survival
rate of patients and emerged as an independent prognostic factor. The
downregulation of the Drg-1 gene appeared to be largely at the RNA
level, and the DNA methylation inhibitor, 5-Azacytidine, significantly
elevated the Drg-1 gene expression in various breast tumor cell lines.
Furthermore, they found that overexpression of the Drg-1 gene
suppresses the invasiveness of breast cancer cells in vitro, and this
suppression was also achieved by treatment of cells with 5-Azacytidine
(139). Moreover, combination of the two markers, PTEN and Drg-1,
of the most aggressive forms of breast cancer, inflammatory breast cancer
(IBC) and invasive micropapillary carcinoma (IMPC), often overexpress
E-cadherin (135–137). Thus, evaluating E-cadherin expression alone in
breast cancers is more useful for distinguishing lobular from ductal
carcinomas than predicting clinical outcome.
upregulates the tumor metastasis suppressor gene Drg-1 in breast cancer
(138). Further studies by the same group demonstrated that PTEN
emerged as a significantly better predictor of prostate and breast cancer
patient survival than either marker alone. Thus these results strongly
suggest functional involvement of the Drg-1 gene in suppressing the
metastatic advancement of human breast cancer.
3.2.9. TIMP
Breast cancer cells need to cross the basement membrane (BM) tissue
boundaries. MMPs are enzymes with proteolytic activity towards
extracellular matrix components (ECM) of the BM, which are blocked
by physiological tissue inhibitors of metalloproteinases (TIMPs). Cancer
metastasis occurs as a result of an imbalance between MMPs, and their
inhibitors. In cultured breast cancer cell lines, transfection of TIMP-4
into the invasive human breast cancer cell line MDA-MB-435 reduced
invasion in an in vitro model system (29, 140) and overexpression of
TIMP-2 in MDA-231 cells reduced osteolytic lesions after injection of
these cells into nude mice (141). Giannelli et al. found that pro-MMP-9
and TIMP-1 serum concentrations are inversely correlated in breast
cancer patients. Their results show that after surgery, when the breast
cancer tissue was removed, pro-MMP-9 concentrations dramatically
in primary breast carcinomas are associated with development of distant
decreased and TIMP-1 concentrations strongly increased (142). Ree et al.
showed that high levels of messenger RNAs for TIMP-1 and TIMP-2
20 Samant, Fodstad, and Shevde
infiltrative breast carcinomas, showed a correlation of TIMP-2 with
proliferative activity and patient survival in breast cancer (142). It is
carcinoma.
3.2.10. RKIP
RKIP was described as a physiologic endogenous inhibitor protein of
the Raf-1/mitogen-activated protein kinase (MAPK) kinase/extracellular
signal-regulated kinase (ERK) pathway. RKIP interferes with the Raf-1-
mediated phosphorylation and activation of MAPK kinase via its ability
to disrupt the interaction between the two kinases. Treatment with
chemotherapeutic agents induces RKIP expression, sensitizing the breast
and prostate cancer cells to apoptosis. This is corroborated by a similar
effect of ectopic expression of RKIP in breast cancer cells that are
endogenous RKIP by expression of antisense and small interfering RNA
(siRNA) confers resistance on sensitive cancer cells to anticancer druginduced
apoptosis. In a large clinical cohort comprising 103 patients with
metastatic and nonmetastatic breast cancer, RKIP expression was high in
breast duct epithelia and retained to varying degrees in primary breast
tumors. However, in lymph node metastases, RKIP expression was highly
by tumor tissues may be a determinant of the progression in breast
possible, that the imbalance between MMPs and TIMPs produced
resistant to the effects of DNA-damaging agent. This sensitization can
be reversed by upregulation of survival pathways. Downregulation of
significantly reduced or lost (143, 144). RKIP expression is independent
of other markers for breast cancer progression and prognosis.

BREAST CANCER METASTASIS CONTROLLING GENES

These are the proteins that are implicated to influence critical steps in
the resulting in promotion of metastasis.
These critical steps and the genes involved are summarized in Table 1.
3.1.1. Immune evasion
Cancer cells can grow by escaping from the attack of immune cells,
thus disrupting the host immune system, which is progressively suppressed
as a result of tumor progression and metastasis. The molecular
mechanisms by which cancer cells evade the host immune system have
been investigated in mouse models and clinical samples.
Tumor cells employ several mechanisms to evade immune response
including loss of tumor antigen, alteration of HLA class I antigen, defective
death receptor signaling, lack of costimulation, immunosuppressive
cytokines, and immunosuppressive T cells (9). Gutierrez et al. showed
that FasL expression by breast tumor plays a central role in the induction
Genetic control of breast cancer metastasis 9

phosphorylation, glycosylation, acetylation, etc. have significant contri-
3.1. Metastasis-Promoting Genes (MPGs)
discernable using xenograft studies or mouse mammary tumor model
studies. On the other hand search for metastasis suppressing genes had
started in mid- to late 1980s and the field really flourished at the turn of
the millennium (7, 8).
lymphocyte apoptosis and impairs expression of NKG2D and T-cell
activation. A study by Ueno et al. reports that compared with healthy
female controls, breast cancer patients, especially those with liver
metastases, have higher circulating sFas levels (13).
Table 1. Critical steps and genes involved in breast cancer metastasis
Steps in breast cancer
metastasis
Genes involved
1 Immune evasion Fas and FasL
2 Adhesion Selectins, integrins, lectins, and cadherins
3 Invasion (proteolysis) Metalloproteinases, uPA, serine
proteinases, and cathepsins.
4 Motility Autotaxin, and hepatocyte growth factor
(HGF)
5 Chemo attractants (tumor
environment)
Osteonectin (SPARC), CXCR4, and
CCR7
6 Cytoskeletal rearrangement S100A4
7 Cell survival Osteopontin
8 Gene regulation (chromatin
remodeling)
MTA1
9 Molecules with mechanisms COM1, RKIP
3.1.2. Adhesion
Metastatic cells need to detach from the primary site and attach at the
secondary site. Thus it needs an intricate expression control of various
adhesion molecules on the cell surface in space and time (14). Specific
families of adhesion molecules whose expression correlates with metastasis
include selectins, integrins, lectins, and cadherins. Details about
these molecules have been discussed by Shevde and King in chapter 6.
3.1.3. Invasion (Proteolysis)
The degradation of the extracellular matrix is mediated by a number
of families of extracellular proteinases. These families include the serine
proteinases, such as the plasminogen-urokinase plasminogen activator
like cathepsin D and L (24–27), and the zinc-dependent matrix metalloproteinases
(MMPs). There are many observations from various research
groups highlighting the central role of MMP-driven extracellular matrix
10 Samant, Fodstad, and Shevde
yet to be confirmed
(uPA) (15,16) and leukocyte elastases (17–23), the cysteine proteinases,
of apoptosis of infiltrating Fas-immune cells providing a mechanism for
tumor immune privilege (10). It was also observed that FasL in breast
tissue is functionally active and that tamoxifen inhibits FasL expression,
allowing the killing of cancer cells by activated lymphocytes (11). Fas
exists in two forms, transmembrane and soluble (sFas). A study by Bewick
et al. suggests that plasma levels of sFas may be a valuable clinical prognostic
factor in predicting outcome for patients with metastatic breast
cancer undergoing high-dose chemotherapy (12). sFas induces host
3.1.4. Motility
There are several secreted signals that decide motility in cancers. One
of the key factors that affect motility is the autocrine motility factor,
autotaxin.
Autotaxin
Autotaxin (ATX) is a novel metastasis-enhancing motogen and angiogenesis
factor. Yang et al. found that the expression of ATX mRNA was
closely linked to invasiveness of breast cancer. This was supported by
immunohistochemical analysis of the breast tissues. MDA-MB-435S
breast cancer cells, that express higher amount of ATX mRNA, show
greater relative invasiveness to fibroblast-conditioned medium than
MCF7, MDA-MB-231, and HBL-100 breast cancer cells. Furthermore,
ATX-transfected MCF7 cells showed increased motility and invasiveness
compared to vector-transfected MCF7 cells (34).
Hepatocyte growth factor (HGF) or scatter factor (SF)
Hepatocyte growth factor (HGF) has been reported as the cause of
many biological events, including cell proliferation, movement, invasiveness,
morphogenesis, and angiogenesis. Sheen-Chen et al. reported that
breast cancer patients with more advanced TNM staging were shown to
have higher serum soluble HGF. Thus, preoperative serum soluble HGF
levels might reflect the severity of invasive breast cancer (35). This is supported
by a paper by Taniguchi et al. that reports a significant increase in
the circulating level of HGF in primary breast cancer patients as compared
to healthy controls. Additionally, 82.9% patients with recurrent
breast cancer had an increase in the serum HGF level (36). Yamashita
et al. measured immunoreactive (ir)-HGF concentration in tumor extracts
of 258 primary human breast cancers and found that breast cancer
patients with high ir-HGF concentration had a significantly shorter
relapse-free and overall survival rate when compared to those with low
ir-HGF concentration. Thus hepatocyte growth factor is a strong and
independent predictor of recurrence and survival in human breast cancer
(37). There are several cell line and animal model studies that support
2. Genetic control of breast cancer metastasis 11
cancer dissemination. High levels of two MMPs (i.e., MMP-2 and stromelysin-
3) have been found to correlate with poor outcome in patients
with breast cancer, (28–30). Batimastat reduced both lung colonization
and spontaneous metastasis of a highly malignant rat mammary cancer
by antisense oligodeoxynucleotides prevented invasion of an artificial
(31). In mouse mammary cancer cell lines, inhibition of stromelysin-1
remodeling in mammary gland development, breast cancer, and breast
basement membrane (32). The ratio of active to latent form of MMP-2
increased with tumor progression in invasive breast cancers (33).
This resulted in increased adhesion of tumor cell lines to bone marrowderived
endothelial cells and transendothelial migration of cancer cells
(41). Martin et al. showed that HGF decreased transepithelial resistance
and increased paracellular permeability of two human breast cancer cell
lines, MDA-MB-231 and MCF7. HGF modulates the levels of several
tight junction molecules including occludin, claudin-1 and -5, JAM-1
and -2 in these cells. Thus, HGF disrupts tight junction function in
human breast cancer cells by effecting changes in the expression of tight
junction molecules (42). Using multiple approaches including ribozymes
serine protease inhibitors of HGF activity (43), the Jiang laboratory has
demonstrated that HGF plays a crucial role in cancer metastasis (48).
3.1.5. Chemo attractants (Tumor environment)
Osteonectin
12 Samant, Fodstad, and Shevde
(43, 44), NK4 (a variant form of HGF) (45-47), and novel Kunitz-type
SPARC (secreted protein acidic and rich in cysteine), also known as
osteonectin is a secreted glycoprotein which is detected in a number of
normal and neoplastic human tissues in vivo. It is an extracellular matrix
(ECM)-associated protein which is postulated to regulate cell migration,
adhesion, proliferation, and matrix mineralization. Early studies by
Graham et al. report that loss of ER expression may lead to overexpression
of osteonectin and contribute to a poorer differentiated, more invasive
phenotype (49). SPARC is also reported to decrease levels of TIMP-2,
causing an increase in the activation of MMP-2 in breast cancer cells
(50). Additionally, osteonectin is indirectly controlled by c-Jun and can
increase invasion and motility of MCF7 breast cancer cells (51). Campo
McKnight et al. showed that osteonectin isolated from conditioned media
of several breast cancer cell lines enhances the migration of breast
cancer cells to vitronectin (52). Jacob et al. showed that the purified active
factor from bone and from marrow stromal-cell-conditioned medium is a
low glycosylated osteonectin that specifically promotes the invasive
this patient data. HGF stimulates tumor growth and tumor angiogenesis
of human breast cancers in the mammary fat pads of athymic nude mice
(38) and also promotes spontaneous metastasis of human metastatic
breast carcinoma MDA-MB-435 cells (39). Mechanistic insight about
HGF was developed when Matteucci et al. reported that HGF enhanced
CXCR4 mRNA and protein expression in MCF7 (low invasive)
carcinoma cells; while in response to hypoxia, CXCR4 induction was
observed in both MCF7 and MDA-MB-231 (highly invasive) carcinoma
cells. Thus HGF and hypoxia may contribute to breast carcinoma cell
invasiveness by inducing the chemokine receptor CXCR4 (40). Studies
by Mine et al. demonstrated that HGF stimulated breast cancer cells by
upregulating CD44 expression via the tyrosine kinase signaling pathway.
in breast cancer and as such has a significant bearing on patient
prognosis and long-term survival.
Chemokine receptors
Chemokine receptors are defined by their ability to induce directional
migration of cells toward a gradient of a chemotactic cytokine
(chemotaxis). In particular, the chemokine CXCL12 and its receptor
CXCR4 have prominent roles in primary and metastatic breast cancer
(56, 57). Binding of CXCL12 to CXCR4 induced activation of the Akt
pathway, MAPK pathway, and the Jak-Stat pathway, culminating in
increased motility, invasion, and survival (58). Abrogating expression of
CXCR4 and CXCR3 functionally inhibits growth and metastasis of
breast cancer in murine models (59). The clinical significance of CXCR4
in breast cancer is widely reported. CXCR4 associated with increased
risk of metastasis to the liver (60–62), CXCR1 was associated with
metastasis to the brain (60–62). Patients with chemokine receptor CCR6
positivity were more likely to develop a first metastasis in the pleura. In
addition, chemokine receptor CCR7 expression was associated with the
occurrence of skin metastases (61). Thus expression of chemokine
receptors in the primary tumor predicts the site of metastatic relapse in
that expression of CXCR4 is associated with axillary lymph node status
in patients with early breast cancer (63). Similar findings were also
role in the breast cancer metastasis.
S100A4
The calcium-binding S100A4 protein has been associated with increased
metastatic capacity of cancer cells, and recent studies have suggested an
2. Genetic control of breast cancer metastasis 13
patients with axillary node positive breast cancer. Su et al. demonstrated
3.1.6. Cytoskeleton rearrangement
reported by Kang et al. (64). Thus chemokine receptors play a deciding
In clinical specimens, high expression of osteonectin in breast tumor
tissues was seen in ductal as well as lobular tumors. Increased expression
of osteonectin was seen in Grade 3 and TNM2 and TNM4 tumors. Nodepositive
tumors also exhibited higher levels of SPARC than nodenegative
tumors. It was also noted that SPARC was present in high
ability of bone-metastasizing breast cancer cells but not that of nonbonemetastasizing
tumor cells (53). These reports are contrasted by a study by
Dhanesuan et al. who conclude that SPARC, in fact, is inhibitory to human
breast cancer cell proliferation, and does not stimulate migration (54).
levels in NPI2 and NPI3 tumors. Over a 6-year follow-up period, high
levels of SPARC was seen to be significantly associated with the overall
correlation with disease-free survival (55). Thus, overall, SPARC
appears to play a crucial role in tumor development and aggressiveness
survival of the patients (P=0.0198). However, there was no significant
3.1.7. Cell survival
Osteopontin
Osteopontin (OPN) is a secreted, integrin-binding phosphoprotein that
is produced by a limited number of normal tissues, including bone and
other mineralized tissues. OPN expression specifically within the tumor
detected in the plasma of late-stage breast cancer patients (74, 75). Since
OPN is expressed by both tumor infiltrating lymphocytes as well as the
tumor cells themselves, OPN expression specifically within the tumor
cells correlates with patient survival (73).
OPN signaling acts to enhance malignancy by giving the cells a
survival/growth advantage. OPN also augments attributes that confer
increased aggressiveness by activating expression of genes and functions
that contribute to metastasis. In concert with growth factor receptor
pathways, such as EGFR and c-met, OPN can accentuate effects of EGF
and HGF/scatter factor respectively (76, 77). A recent study reports that
OPN induces multiple changes in gene expression that reflect the six
sufficiency in growth signals, insensitivity to antigrowth signals, evading
apoptosis, tissue invasion and metastasis, sustained limitless replicative
enhanced incidence of bone metastases by breast cancer cells with
combined overexpression of OPN and interleukin-11, which could be
further increased by the overexpression of CTGF (79). Moreover, a specific
splice variant of OPN is associated with conferring an aggressive
phenotype upon breast cancer cells (80). Thus, in a nutshell, OPN potentiates
the attributes of tumor cell survival and aggressiveness.
‘‘hallmarks of cancer’’ in a model of breast cancer progression: selfcells
reciprocally correlated with patient survival (72, 73). OPN is
growth genetic instability, and angiogenesis (78). Kang et al. showed
inverse correlation between the expression level of S100A4 and survival
of breast cancer patients (65, 66). Functionally, the introduction of
S100A4 into MCF7 cells enables the MCF7 cells to grow tumors in mice
in the absence of estrogen, i.e., S100A4 confers estrogen-independence
upon the breast cancer cells (67). The C-terminal region of S100A4 is
important for its metastasis-inducing properties, deletion of the last 15
nonmalignant tumors in neu transgenic mice and in malignant tumors
from neu/S100A4 double transgenic mice (69). Clinically, S100A4
expression is an indicator of a poor prognosis for T1N0M0 breast cancer
(70). High levels of S100A4 expression in combination with either Met
or OPN correlate with adverse prognosis and low survival (70, 71).
While there is no single mechanism attributed to S100A4 to increase
aggressiveness of cancer cells, the increased levels are undisputedly
amino acids of S100A4 reduced motility/invasion (68). S100A4 regulates
cell motility and invasion in epithelial cells lines isolated from
14 Samant, Fodstad, and Shevde
associated with higher grade of the tumor and poor prognosis.
loss of E-cadherin and decreased cytoplasmic beta-catenin. MTA2
expression is correlated with ERalpha protein expression in invasive
breast tumors (87). MTA2 binds to ERalpha and represses its activity
in human breast cancer cells. Furthermore, MTA2 inhibits ERalphamediated
colony formation and renders breast cancer cells resistant to
estradiol and the growth-inhibitory effects of the antiestrogen tamoxifen
(88). Recent studies have also shown that growth factor stimulation of
breast cancer cells induces the expression of MTA1 and its interaction
with and repression of the estrogen receptor (ER) transactivation funchormone
independence. Furthermore, the status of the ER pathway modulates
the expression of MTA3 as well as epithelial-to-mesenchymal
transition in human breast tumors (81, 89)