Introduction
Breast cancer is the most common cancer in women
worldwide. Triple-negative breast cancer (TNBC) is
the most aggressive and invasive type of breast cancer
with poor prognosis. Anthracyclines-based combination
chemotherapy is the standard treatment for patients with TNBC.[] Targeted treatments essentially
improve the patient's outcome for some types of
breast cancer. However, in particular, TNBC has faced
some potential challenges. Novel treatment strategies
for TNBC use have targeted therapies in combination
with apoptotic ligands, such as TRAIL and chemotherapy.
However, the recurrence and metastasis of TNBC due to chemoresistance takes place in up to 70% of the
patients.[]
Metastases account for 90% of human cancer
deaths. In cancer, metastasis and resistance to chemotherapy
are linked phenomena.[] Especially in breast
cancer, metastasis is a major cause of fatality. Bone,
lung and liver are the main sites of metastases in this
disease.[] Chemoresistance often hampers tumor cells
from undergoing sufficient levels of apoptosis, resulting
in cancer cell survival and treatment failure.[]
Chemotherapy, as well as targeted therapies, is aimed
to reduce cell growth, survival, and metastasis and inducing
apoptosis in breast cancer cells. However, their
effectiveness is limited to the development of resistance
and tumor progression. The molecular mechanisms
that lead to therapeutic resistance are diverse and often
involve apoptosis at various levels in signal cascades
and are still incompletely understood.[]
The apoptosis or programmed cell death is considered
as an important homeostatic mechanism that
equilibrates cell generation with cell death and maintains
correct cell numbers in the body in physiological
and pathological conditions.[] Two fundamentally
distinct apoptotic signaling pathways have been determined
in mammalian cells: extrinsic (or death receptor
pathway) and intrinsic (or mitochondrial) pathways.
[] The extrinsic pathway involves the activation of
death receptors (DRs). The DRs are members of the tumor
necrosis factor receptor superfamily and include
a subfamily that is characterized by an intracellular
death domain. Among all the DRs, DR-4 and DR-5 are
selectively expressed in cancer cells and, thus, offer an
advantage for targeted therapy and prevention.[,]
In the metastatic cascade, certain tumor cells detach
from the primary tumor, circulate in the bloodstream
and finally exit from the blood and constitute a new tumor
inappropriate tissue. Tumor cells in different steps
of these processes have distinctive and appropriative
properties that will help them during this process.[]
Identification of these specific features could be
useful in the designing of new therapies. Several studies
have focused on the molecular level of the metastasis
process.[,] Metastatic cells tend to exhibit a
higher survival ability and resistance to apoptosis and
become resistant to treatment. It is postulated that the
increase in apoptotic resistance of metastatic cancer
cells is associated with modifying expression levels or
function of proteins involved in apoptosis signaling
pathways.[]
There is little direct experimental evidence linking
apoptosis and metastasis, the apoptotic resistance of metastatic cells were hence the focus of this study. We
created a lung metastatic mice model of TNBC. Primary
and metastatic tumor cells were generated and
the expression of the DR5 as an execution factor of
apoptosis was compared between these cells.
Methods
Cell Culture
4T1 cell line was purchased from the cell bank of Pasteur
Institute of Iran. The cells were cultured in high
glucose Dulbecco"s Modified Eagle"s Medium (DMEM)
containing 10% FBS (fetal bovine serum) and 2% Penicillin-
Streptomycin (all from Gibco, USA) in humidified
atmosphere and 5% CO2 at 37 C.
Induction of Syngeneic Animal Model of Breast
Cancer
Female BALB/c mice weighing 20 to 25 gram was obtained
from Royan Institute of Iran. The animals were
housed in cages at 12-h photoperiod and free access
to food and water. All animal experiments were in
compliance with the relevant laws and were approved
by the Ethics Committee of the Tehran University of
Medical Sciences (registration number: IR.TUMS.
REC.1394.1439). 4T1 cells were subcutaneously injected
to the flank (OR the right hind limb) of the mice
(105 cells suspended in 100 ?L PBS) using an insulin
syringe with a 32g needle. The mice were monitored
daily for appearance and behavior characteristics.
Lung Metastatic and Primary Breast Tumor Cell
Extraction
For extraction of the primary and metastatic tumor
cells, after 35 days of tumor induction in mice, primary
tumor and lung of cancerous mice were excised
and surface blood was removed by rinsing in PBS.
After mincing with scissors, fragments were placed
into a 50 ml conical tube. For enzymatic digestion,
the primary tumor and the lungs were digested in 10
mg?ml collagenase type IV at 37°C for 75 min on a
platform rocker. All enzymes were purchased from
Sigma (St Louis, MO, USA). The digested organ, filtered
through 70-um cell strainers and washed twice
with PBS. Cells were resuspended in medium containing
10% FBS and 2% Penicillin-Streptomycin
and cultured in a cell culture flask in the humidified
atmosphere and 5% CO2 at 37°C. The metastatic tumor
cells in the lung, after primary isolation, form
colonies in the culture medium. Due to the high rate
of growth and proliferation, the tumor cells in these colonies are purified after several passages. These
metastatic tumor cells are called 4T1L, while tumor
cells obtained in the same way as the original tissue of
the tumor called 4T1P.
Quantification of DR5 by RT-qPCR
The real-time PCR procedure was executed based on
the 1 µg/µ?l cDNA in all samples. The primary and
lung metastatic mammary tumor cells (4T1P and
4T1L) were grown in 6 cm Petri dishes to 80% confluence.
Total RNA was extracted from these cells using
QIAzol Lysis Reagent-QIAGEN. The RNA quality,
yield, and size of extracted RNA were analyzed using
nanodrop and electrophoresis. The first-strand cDNA
synthesis was performed with the reverse transcription
system (PrimeScript™ RT reagent Kit (Perfect Real
Time) Takara). Quantization of all gene transcripts
was done by real-time quantitative PCR using Power
SYBR Green PCR Master Mix and an RT-qPCR analysis
for DR-5 was carried out with SYBR Green Realtime
PCR Master Mix (Amplicon A/S, Denmark) according
to the respective manufacturer!s instruction.
The exact mRNA expression was normalized to the
expression level of GAPDH. Relative changes in gene
expression were calculated by the following formula,
and the data were represented as fold up-regulation/
down-regulation.
Fold change=2-ΔΔCt, where ΔΔCt=[Ct of gene of
DR (in 4T1L)-Ct of gene GAPDH (in 4T1L)]-[Ct of
gene of DR (in 4T1P)-Ct of gene GAPDH (in 4T1P)]
The primers used were as follows: for DR5, forward
5?-AAAACGGCTTGGGCATCTTGGC-3'; reverse5'-
AGACGGTTCCAGGAGTCAAAGG-3'; for GADPH,
forward 5'-GGTGAAGGTCGGTGTGAACG-3'; reverse
5'-CTCGCTCCTGGAAGATGGTG-3'.
RNA and DNA Gel Electrophoresis Assay
Total extracted RNA from treated cells and real-time
PCR, DNA products were electrophoresed on 1.2%
agarose gel containing 0.5 ug/ml safe stain(EB) (Sigma-
Aldrich, MO, USA) for 1 h. DNA and RNA ladders
were visualized and photographed under the transmitted
ultraviolet light.
Statistical Analysis
Results were expressed as the mean±standard deviation.
Data were analyzed using GraphPad Prism statistical
software 6.0 (GraphPad Software, La Jolla, CA,
USA) using either Paired Samples t-Test or two-way
ANOVA where appropriate. P<0.05 was considered
statistically significant.
Results
Induction of Syngeneic Animal Model of Breast
Cancer
Metastatic animal model of triple-negative breast cancer
was generated after 35 days of tumor induction in
Balb/c mice (Fig. 1a). H&E staining and pathological
confirmation were performed on tumor tissues (Fig. 1
b, c, d, and e).
Fig 1: Primary and lung metastatic tumor cells extraction. (a) Metastatic animal model of triple-negative breast cancer
was generated after 35 days of tumor induction in Balb/c mice. (b, c) primary tumor and lung of cancerous mice
removed. (d, e) H&E staining and pathological confirmation were performed on tumor tissues. (f, g) Primary and
metastatic tumor cells were extracted from subcutaneous tumor and lung of cancerous mice, respectively.
Primary and Metastatic Tumor Cells Extraction
We properly extracted primary and lung metastatic tumor
cells from subcutaneous primary tumor and lung
of cancerous mice, respectively. The metastatic tumor
cells in the lung, after primary isolation, form colonies
in the culture medium. Due to the high rate of growth
and proliferation, the tumor cells in these colonies are
purified after several passages. These metastatic tumor
cells are called 4T1L, while tumor cells that are
obtained in the same way as the original tissue of the
tumor called 4T1P (Fig. 1f, g).
Down-regulation of DR-5 in Metastatic Tumor Cells
Apoptotic cell death is related to the activation of regulatory
genes. Therefore, the expression level of its related
genes was measured. The expression of DR-5 as
an execution factor of apoptosis was analyzed in 4T1L
and 4T1P. Total RNA extraction, cDNA synthesis, and
real-time qPCR were done as described in material
and methods. The quality, yield, and size of extracted
RNA, synthesized cDNA and PCR products were
confirmed using nanodrop and gel electrophoresis
(Fig. 2a, b). The expression of DR-5 was significantly
down-regulated in 4T1L compared with 4T1P (Fig.
2c). These results suggest that DR-5 has a significant
role in the apoptosis resistance of metastatic tumor
cells.
Fig 2: Down-regulation of DR-5 in 4T1L confirmed by
real-time PCR. (a, b) The quality and size of extracted
RNA and PCR products were confirmed
using gel electrophoresis. (c) The DR-5 gene
expression was significantly down-regulated in
metastatic tumor cells. All results are expressed
by mean±SD from at least three independent
experiments analyzed by the Two-tailed T-test.
**P<0.001. Down-regulation of DR-5 in 4T1L
confirmed by real-time PCR. (a, b) The quality
and size of extracted RNA and PCR products
were confirmed using gel electrophoresis. (c) The
DR-5 gene expression was significantly downregulated
in metastatic tumor cells. All results are
expressed by mean±SD from at least three independent
experiments analyzed by the Two-tailed
T-test. **P<0.001.
Discussion
A decreased sensitivity to apoptosis induction plays
critical roles in tumorigenesis, invasion, metastasis,
and chemoresistance of breast cancers. Many questions
regarding apoptosis resistance in breast cancer
remain to be answered.[] Apoptosis, a form of programmed
cell death, requires the cascaded activation
of caspases. Activation of procaspases resulted from
two major pathways, intrinsic and extrinsic. The extrinsic
pathway is associated with death receptors.[]
Among the six different death receptors (DRs) identified
to date, DR-4 and DR-5 are selectively expressed on cancer cells. Therefore, unlike chemotherapeutic
agents, these receptors can potentially mediate the selective
killing of tumor cells.[] DR-5 is well known
to mediate apoptosis upon ligation with its ligand or
induction of its clustering or aggregation (e.g., with
an agonistic antibody or overexpression). Targeting
the DR-5 extrinsic apoptotic pathway has become an
attractive cancer treatment strategy.[] It has been
documented that metastatic cells are more resistant to
apoptosis.[] However, to date, the precise mechanism
of this resistance has not been clearly addressed. To our
knowledge, in this study, DR-5 gene expression in the metastatic tumor cells and their ancestors, the primary
tumor cells were reviewed for the first time. Results
showed that, compared with the primary tumor cells,
DR-5 gene expression was significantly down-regulated
in metastatic tumor cells.
Current research in TNBC, which is an aggressive
subtype of breast cancer, is focused on a greater understanding
of the response and resistance to treatment,
including the role of apoptosis.[] Despite a variety
of available approaches for the treatment of metastatic
TNBC, the survival rate of patients remains very low. In
TNBC, metastasis and chemo-resistance are the most common
causes of treatment failure. Elucidating the
underlying mechanisms is crucial to developing novel
therapeutic strategies.[] The result of the present
study elucidates that DR-5 gene expression down-regulated
in the metastatic cascade of TNBC and this can
be a major reason for apoptotic resistance of metastatic
tumor cells.
It is unclear whether DR-5 indeed plays a role in
the regulation of cancer invasion and metastasis in
humans. In some human cancer, such as breast, lung
or renal cell cancer, results indicate that DR5 is overexpressed
and significantly correlated with more aggressive tumor behavior and poor survival of cancer
patients.[-] In other cancer (e.g., in the bladder or
colorectal cancer) results show that DR-5 expression is
associated with a less aggressive phenotype and better
survival.[,] In other cancer (e.g., in the bladder or
colorectal cancer) results show that DR-5 expression is
associated with a less aggressive phenotype and better
survival.[,]
Our result showed that DR-5 down-regulated in the
metastatic cascade of TNBC. In compliance with our
results, research on human cancer cell lines showed
that inhibition of DR-5 by knockdown or knockout increased
invasion of cell lines. Strikingly, knockdown of
DR-5 expression significantly increased lung metastasis
of cancer cells in a nude mouse subcutaneous lung
cancer xenograft model.[]
DR-5 Mutations have been identified in various human
tumors.[-] In breast cancer, approximately
12% of inactivating mutations in DR-5 was diagnosed
only in metastatic type.[] It has been shown that DR-5
deficiency in mice enhances lymph node metastasis of
skin carcinoma [] and metastasis of lymphoma cells
to liver and lung.[] It has been shown that the DR-5
agonistic antibody lexatumumab robustly suppresses
lymph node or lung metastasis in an orthotopic model
of TNBC.[]
Conclusion
In conclusion, the results of this research demonstrated
that compared with the primary tumor cells, DR-5
gene expression was significantly down-regulated in
metastatic tumor cells, and consequently, these cells are
more prone to apoptotic resistance. These findings support
the notion that DR-5 may be associated with the
suppression of cancer metastasis and down-regulation
of this receptor in tumor cells strategies to invade and
metastasis. Consequently, the findings obtained in this
study will highly likely to provide the basis for designing
targeted therapeutic methods to combat metastatic
TNBC.
Acknowledgments: We want to especially thank Dr. Naser
Ahmadbeigi for his practical advisation and helpful information
about metastatic breast cancer.
Peer-review: Externally peer-reviewed.
Conflict of Interest: The author declares that they have no
competing interests.
Ethics Committee Approval: All animal experiments
were in compliance with the relevant laws and were approved by the Ethics Committee of the Tehran University
of Medical Sciences (Registration number: IR.TUMS.
REC.1394.1439).
Financial Support: This work was supported by a grant
from the Tehran University of Medical Sciences (TUMS)
Grant No 95-03-87-33016.
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