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.