Introduction
Breast cancer is the most diagnosed cancer in female
gender worldwide and the primary cause of cancer
deaths in women.[] Breast cancer generally develops from hyperproliferation of ductal cells and then evolves
into benign or metastatic tumors. There are multiple
risk and causative factors for breast cancer development
such as aging, smoking, reproductive factors such as
early menstruation, late menopause, and family history. [] In addition to the role of tumor microenvironment,
many genes have been associated in the pathogenesis of
the disease.[] Mutations and/or abnormal amplification
of these genes have been related to tumor initiation
and progression. Breast cancer is categorized into 3
subtypes based on the presence or absence of molecular
markers.[] Among the molecular markers responsible
for breast cancer development, estrogen receptor alpha
(ER) expression is found nearly 70% of the invasive
breast cancer cases.[] ER is a steroid hormone receptor
(HR) and when is induced by estrogen, activates downstream
oncogenic signaling pathways. In addition to ER
expression, progesterone receptor (PR) is also a marker
of ER signaling. Tumors expressing either ER or PR are
categorized as HR positive and primary therapy method
is the use of endocrine agents. The second category of
breast cancer is defined with the positivity of epidermal
growth factor 2 (ERBB2, previously HER2). The third
category of breast cancer is called triple-negative tumors
which are characterized by the lack of expression of molecular
markers mentioned above ER, PR, or ERBB2.
Triple-negative breast cancer makes up nearly 15% of
all types and has a high risk of relapse within the 3-5
years following diagnosis.[] As majority of the breast
tumors are HR+, the main anti-cancer agent group used
in the therapy is selective estrogen receptor modulators
(SERMs). Tamoxifen is a SERM and inhibits estrogen
hormone from entering cells, therefore inhibits breast
cancer cell development.[] 5 years" tamoxifen treatment
of patients with HR+ breast cancer reduces recurrence
rate 30-50% depending on the anatomic stage of
the disease.[] Thus, clinical studies have evaluated the
longer use tamoxifen, but these studies showed a small
improvement in the recurrence rate.[] Furthermore,
large number of patients develops resistance to tamoxifen
in years.[] Tamoxifen resistance has been related
to number of genes, other molecular factors, and activation
or downregulation of signaling various signaling
pathways.[,] Benefits from the studies investigating
resistance factors to clinical use are highly limited.
Cholesterol is a non-essential metabolite required
for normal cell growth and proliferation. For new
membranes to be made in proliferating cells and for
the cell signal transmission system to continue actively,
endogenous cholesterol synthesis and the intake
of lipid particles from the outside are needed.[]
Altered cholesterol metabolism has been reported in
various cancer cells.[,]
Transmembrane protein 97 (TMEM97), also known
as MAC30, is member of TMEM proteins that span the
lipid bilayer of membranes such as plasma membrane and organelle membranes. Recently, TMEM97 was
identified as the sigma receptor 2 which acts as opiate
receptor and gained more attention.[] Furthermore,
TMEM97 has been related to cholesterol metabolism as
it has been shown to interact with LDLR.[] TMEM97
gene expression varies among cancer types. In pancreatic
and prostate cancers, low TMEM97 expression has been
reported.[] On the other side, high TMEM97 expression
has been found in breast, gastric, colon, ovarian, oral
squamous, and non-small cell lung cancer cells, and thus,
it is defined as presumptive oncogene.[] Ro48-8071 is
a cholesterol-lowering agent by inhibiting oxidosqualene
cyclase, an enzyme in the cholesterol biosynthesis steps.
Ro48-8071 and tamoxifen have been shown to have higher
affinity for TMEM97 binding suggesting the protein as
pharmacologically druggable.[] This data highlight the
possible role of TMEM97 in cholesterol metabolism.
Identifying new drug targets could be beneficial in
understanding of the molecular basis of tamoxifen resistance.
Based on these observations in the literature,
this project was aimed to investigate whether TMEM97
could be a target for tamoxifen.
Methods
Cell Culture and Medium
MCF7, MDA-MB-231, and MCF10A cell lines are kind
gifts by Dr. Ceren SARI of Karadeniz Technical University,
Institute of Health Sciences. All cell lines except
from MCF10A were cultured in DMEM medium
(Gibco, ThermoFisher,) supplemented with 10% FBS,
1% pen/strep, and 1% l-glutamine. MCF10A cells were
cultured in DMEM/ Ham's F-12 (Gibco, ThermoFisher)
supplemented with 100 ng/ml cholera toxin, 20 ng/
mL epidermal growth factor, 0.01 mg/mL insulin, 500
ng/mL hydrocortisone, and 5% chelex-treated horse
serum at 37°C with 5% CO2. MCF7/TAMR-1 (ATCC,
CRL-3435) cell line was cultured in phenol red-free
DMEM medium (ThermoFisher cat no: 11054-020)
supplemented with 10% FBS, 1% pen/strep, 1% nonessential
amino acids, and 0.1% insulin (Sigma cat no:
I0516-5ML). When first thawed, the cell line was exposed
to 5 µM tamoxifen (Sigma-Aldrich 4-Hydroxytamoxifen
cat no: T176) 2-3 times.
Generating TMEM97 Knockout MCF7 Cells
Two different sets of single guide RNA (sgRNA) for
TMEM97 gene and one set control sgRNA were determined
(Table 1) using UCSC genome browser and crispr.
mit.edu web pages. Annealed oligonucleotides were
cloned into a plentiCRISPR-Cas9 v2 vector using T4 ligase (NEB). Transfection-grade plasmids were used
for viral packaging in Hek293T cells. Cell supernatant
was collected to obtain virus particles which were
then used to transfect MCF7 cells through spinfection
method in the presence of 8 µg/ml polybrene. After antibiotic
selection, knockout clones were selected with
single-cell cloning. When clones reached enough confluency,
TMEM97 expression was evaluated by western
blotting by selecting different clones obtained.
Table 1 sgRNA list
Immunoblotting
Cultured cells were collected by centrifugation to obtain
a cell pellet and rinsed with ice-cold PBS. Protein
isolation from the cell lines was done with RIPA buffer
supplemented with protease inhibitor (Complete,
Roche). The protein concentration in the lysates was
determined with the BCA (bicinchoninic acid, Pierce,
ThermoFisher) kit. After the protein concentrations
were adjusted to 1 mg/mL, the protein loading dye
was added to the final concentration of 1X. The prepared
protein samples were denatured by boiling for
5 min. Denatured protein samples were run on a 12%
tris-glycine gel by SDS-PAGE electrophoresis. After
electrophoresis, the gel was transferred to PDVF
membrane. Samples were treated with appropriate
primary and secondary antibodies. Images were taken
on Azure C300 gel imaging system.
Drug Treatment and Cell Viability
Cells were plated in 96-well plates at a concentration of
15.000 cells/mL, and then, cells were treated with desired
concentrations of tamoxifen. Cells were cultured
at 37°C for 96 h in an incubator at 5% CO2. At the end of
incubation, cell viability was determined using CellTiter
GLO Luminescent Reagent (Promega). Relative fold
change of cell viability was presented as (%) comparing
luminescence measurements to normal medium.
Cholesterol Assay
MCF7 isogenic cells for TMEM97 expression were cultivated
in medium with or without 10 µM tamoxifen
for 96 h. After incubation, the cells were collected and intracellular cholesterol level was determined using the
Amplex Red Cholesterol Assay Kit (Invitrogen) according
to the manufacturer's instruction. Fluorescence was
measured on a multimode spectrophotometer (BioTek
Cytation 5) with excitation at 530 nm and emission detection
at 590 nm. The values of cholesterol level were
calculated from the standard curve. The results were
presented as µg/ml of cholesterol per mg of protein.
Statistical Analysis
Graphs and statistical analysis were done using Graph-
Pad V7. Student's t-test with Welch"s correction was
applied to compare two groups. TCGA Target GTEx
data set was analyzed on Genome Browser Xena.[]
TMEM97 mRNA expression of breast cancer cell lines
was obtained from cancer cell line encyclopedia.[]
All experiments were performed at least two independent
times with similar results. Cell viability assay was
performed with quad technical replicates.
Results
TMEM97 Expression in Breast Cancer Cells
TMEM97 mRNA expression was detected significantly
higher in tumor samples compared to normal tissue
samples as it was analyzed in Genome Browser Xena.
In addition, high TMEM97 mRNA expression (log
fold >10.5) was found associated with poor survival
rate (Fig. 1). Protein expression was evaluated with immunoblotting
in all cell lines. MCF7 cells were found
to have higher expression of TMEM97, compared to
other malignant cell lines and MFC10A cells (Fig. 2a).
Fig. 1. (a) TMEM97 mRNA expression in breast carcinoma and normal breast tissue samples. TCGA GTEx dataset was
analyzed on Genome Browser Xena. A p value <0.0001 was found with one-way ANOVA test. (b) Kaplan-Meier
survival analysis. High TMEM97 expression could be related to decreased survival rate.
TMEM97: Transmembrane protein 97; GTEX: Genotype-Tissue Expression Portal; TCGA: The Cancer Genome Atlas Portal; ANOVA:
Analysis of variance.
Fig. 2. (a) TMEM97 protein expression of MCF7, MDA MB231, and MCF10A cells lines. (b) Cell viability of cell lines
upon tamoxifen treatment. Data presented as mean±standard deviation.
*: p<0.05; **: p<0.01. TMEM97: Transmembrane protein 97.
Tamoxifen Sensitivity of Breast Cancer Cells
MCF7, MDA-MB2331, and MCF10A cells treated with
tamoxifen at increasing concentrations for 3 days. After
treatment, MCF7 cells were determined more sensitive
to tamoxifen at all doses. MCF10A non-tumorigenic
breast cell line was found more resistant to tamoxifen
at 2.5 and 5 µM. However, MDA-MB2331 cells were
detected more resistant after 10 µM tamoxifen dose
compared to MCF10A and MCF7 cells. All these differences
were found statistically significant (Fig. 2b).
TMEM97 mRNA Expression is Lower in Tamoxifen
Resistant MCF7 Cells
To demonstrate the relation between TMEM97 expression
and tamoxifen resistance, TMEM97 mRNA
was evaluated by qRT-PCR in MCF7 cells and MCF7/
TAMR-1 cells. It was found that TMEM97 mRNA expression
is significantly low in tamoxifen-resistant cells
as compared to MCF7 cell line (p<0.01) (Fig. 3).
Fig. 3. TMEM97 mRNA expression in MCF7 and its
tamoxifen-resistant form. Data presented as
mean±standard deviation.
**: p<0.01. TMEM97: Transmembrane protein 97.
TMEM97 Knockout MCF7 Cells are more Resistant
to Tamoxifen
As TMEM97 protein expression was observed higher in
MCF7 cells and this cell line was found more sensitive
to tamoxifen, we further evaluated the role of TMEM97
expression in tamoxifen response by knocking out the
gene using CRISPR/Cas9 genome editing system (Fig.
4a). TMEM97 knockout two isogenic and one control cell
lines were cultured with tamoxifen for 3 days. It was found
that TMEM97 knockout cells were significantly more resistant
compared to control cell line at all doses (Fig. 4b).
Fig. 4. (a) TMEM97 knockout validation by immunoblotting. (b) Cell viability of isogenic cell lines upon tamoxifen treatment.
Data presented as mean±standard deviation.
**: p<0.01; ***: p<0.001; ****: p<0.0001. TMEM97: Transmembrane protein 97; ns: Not significant.
Tamoxifen Treatment Increased Total Cholesterol
Level in all Cells
Knocking out TMEM97 gene slightly decreased total
cholesterol in MCF7 cells. In the control group, the
amount of total cholesterol was 41 µg/mL per mg protein,
whereas in TMEM97 knockout cells, 36 µg/mL
per mg protein was detected (Fig. 5). We also found
that tamoxifen treatment was increased total cholesterol
level in all isogenic cells at a similar trend (almost
doubled). However, the amount of total cholesterol was
still found higher in TMEM97-expressing cells (Fig. 5).
Fig. 5. Total cholesterol measurement of isogenic cell lines.
*: p<0.05; **: p<0.01. ns: Not significant.
Discussion
Breast cancer is one of the most common cancer types
among women, worldwide.[] There are three subgroups
of breast cancer: ER-positive, ERBB2-positive,
and triple-negative breast cancer and ER is expressed in
almost 70% of the patients.[] Tamoxifen is widely used
in the treatment of ER-positive breast cancer patients.
However, many patients are resistant to tamoxifen at the
beginning of the therapy and a greater number of patients
develop resistance to the drug later in the therapy. [] Up to date, some mechanisms have been associated
with tamoxifen resistance in ER+ breast cancer including
polymorphisms of several genes, changes in expression
of genes that have role in ER signaling.[,,] A
recent genome-wide association study identified candidate
pathways that could be responsible for tamoxifen
resistance in ER+ patients.[] SIRT1 was shown to be
important in breast cancer onset and progression by
involving in Akt signaling.[] Recently, together with
SIRT1, high expression of SRC was found in tamoxifen-
resistant cells and related to poor prognosis.[]
In addition, tongue cancer resistance-related protein1
was found to induce tamoxifen resistance by activating
SGK1-signaling pathway, and thus, it was identified as
a novel drug target as it was related to chemo-resistance
in several cancer types including breast cancer cells.[]
In cells, cholesterol levels are dynamically balanced
by uptake, biosynthesis, transport, distribution, esterification,
and export.[] In recent years, cholesterol biosynthesis
pathway has been identified as a mechanism
of estrogen deprivation resistance in ER+ breast cancer.
[] Elevated expression of genes of the cholesterol synthesis
pathway has been shown to be involved in poor
drug response.[] Furthermore, increased expression
of the genes related to lipid metabolism has been determined.[] TMEM97 also known as MAC30 is a transmembrane
protein and its putative role in cholesterol
metabolism has been shown in several studies.[,]
Recently, TMEM97 has been identified as sigma 2 receptor
and suggested to play role in drug response.[]
Furthermore, increased expression of TMEM97 was
found in gastric, lung, breast, and ovarian cancer, and
high expression level was associated with poor prognosis and tumor recurrence.[] All these data suggested
TMEM97 as a drug target for cancer therapy and recently,
an orally active ligand for TMEM97 has been
developed for pancreatic cancer therapy.[] In breast
cancer samples, TMEM97 expression was shown to be
induced by BRCA1 expression.[] On the other side,
downregulation of TMEM97 expression was shown
to be controlled by p53.[] Silencing of TMEM97
expression inhibited cell proliferation of glioma []
and gastric cancer cell lines.[] Reduced expression
of TMEM97 inhibited cell growth and reduced colony
formation of MCF7 cells.[] In addition, downregulation
of TMEM97 inhibited breast cancer cell invasion
through suppression of Wnt/Β catenin and PI3K/Akt
pathways.[] These data suggest that TMEM97 could
be important for tumor growth. However, knockdown
or knockout of TMEM97 did not change proliferation
of HeLa cells.[] In addition, TMEM97 did not mediate
cytotoxicity through sigma 2 ligand in HeLa cells.
[40] In addition, it has been demonstrated that silencing
of TMEM97 through siRNA-mediated knockdown
leads to a decrease in cell proliferation in colorectal cancer
cells, while its overexpression positively affects cell
proliferation.[] This data should be further evaluated
with a more comprehensive approach in a broad range
of cancer types with the use of various known ligands.
In this study, TMEM97 gene expression was found
higher in breast tumor samples compared to normal
breast tissue samples as analyzed with TCGA GTEx dataset.[] Furthermore, we showed that ER+ MCF7 cell
line has higher TMEM97 protein expression compared
to benign breast tumor cell line MCF10A and triplenegative
breast carcinoma cell line MDA-MB-231. Although triple-negative cell lines are presumed to be
tamoxifen resistant, there are multiple studies in the
literature indicating tamoxifen is cytotoxic and induces
apoptosis through different signaling pathways
in breast cancer cells rather than ER signaling.[-]
It was found that MCF7 cells express higher
TMEM97 protein, and they were more sensitive to
tamoxifen treatment. We, therefore, hypothesized that
tamoxifen sensitivity could be related to TMEM97 expression.
To confirm this hypothesis, TMEM97 expression
was controlled in tamoxifen-resistant MCF7 cell
line. Indeed, it was shown that TMEM97 expression is
decreased in tamoxifen-resistant MCF7 cells. To further
demonstrate the effect of TMEM97 expression on
tamoxifen resistance, TMEM97 KO isogenic MCF7 cells
were generated by CRISPR/Cas9 editing. TMEM97 KO
cells were found more resistant to tamoxifen treatment.
One hypothesis regarding to the role of TMEM97 in
tamoxifen resistance is that the protein has a domain
called EXPERA which is found in sterol isomerase protein
family members such as emopamil binding protein
(EBP).[] Tamoxifen is known to inhibit EBP and its
homolog TM6SF.[] Furthermore, tamoxifen was
found among the ligands of TMEM97.[] Therefore, it
could be speculated that tamoxifen creates a signaling
through TMEM97 protein, and if the protein abundance
is low, tamoxifen cytotoxicity is not appliable for cells.
In this study, tamoxifen increased total cholesterol
level in both KO and control cells at a similar ratio.
However, to clarify the role of TMEM97 in cholesterol
metabolism upon tamoxifen treatment, this experiment
should be done in cholesterol/lipid-free media and level
of lipids should be determined at subcellular level. In
a study with T47D cell line, it was reported that 1 uM
tamoxifen treatment increased both free cholesterol and
cholesterol ester level, which was consistent with our
findings.[] Altered lipid metabolism upon tamoxifen
treatment suggests that genes responsible in the metabolism
could be important for tamoxifen resistance.
Conclusion
To conclude, TMEM97 is a new player in cholesterol
metabolism and has been shown to be an important
factor for tumor development in different cancer cells.
In this study, we showed that for the first time, breast
cancer cells that have higher TMEM97 expression are
resistant to tamoxifen. TMEM97 expression could be
an important indicator for tamoxifen sensitivity as
knockout cells are more resistant to the drug. To further confirm our results, clinical studies should be conducted
with high number of patients to obtain more
reliable results regarding the relation of TMEM97 expression
level and tamoxifen resistance.
Acknowledgment: Authors would like thank Prof. Dr.
Berna Demircan Tan (IMU School of Medicine, Department
of Medical Biology) for critical reading the manuscript.
Peer-review: Externally peer-reviewed.
Conflict of Interest: All authors declared no conflict of interest.
Financial Support: This project is funded by The Scientific
and Technological Research Council of Turkey (TÜBİTAK),
grant no: 118S351.
Authorship contributions: Concept - B.Y.; Design - B.Y.;
Supervision - B.Y.; Funding - B.Y.; Materials - B.Y., S.K.A.,
Y.Y.; Data collection and/or processing - B.Y., S.K.A., Y.Y.,
K.U.Ç.; Data analysis and/or interpretation - B.Y., S.K.A.,
Y.Y.; Literature search - B.Y., S.K.A., Y.Y.; Writing - B.Y.,
S.K.A., Y.Y.; Critical review - B.Y.
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