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)