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).