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.