Lee et al. BMC Molecular and Cell Biology(2024) 25:12
https://doi.org/10.1186/s12860-024-00510-y

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Long non-coding RNA SOX2OT in tamoxifenresistant breast cancer
Jeeyeon Lee1,5, Eun-Ae Kim2, Jieun Kang5, Yee Soo Chae3,5, Ho Yong Park1,5, Byeongju Kang1,5, Soo Jung Lee3,5,
In Hee Lee3,5, Ji-Young Park4,5, Nora Jee-young Park4,5 and Jin Hyang Jung1,5*
Abstract
Hormone receptor (HR)-positive breast cancer can become aggressive after developing hormone-treatment
resistance. This study elucidated the role of long non-coding RNA (lncRNA) SOX2OT in tamoxifen-resistant (TAMR)
breast cancer and its potential interplay with the tumor microenvironment (TME). TAMR breast cancer cell lines
TAMR-V and TAMR-H were compared with the luminal type A cell line (MCF-7). LncRNA expression was assessed via
next-generation sequencing, RNA extraction, lncRNA profiling, and quantitative RT-qPCR. SOX2OT overexpression
effects on cell proliferation, migration, and invasion were evaluated using various assays. SOX2OT was consistently
downregulated in TAMR cell lines and TAMR breast cancer tissue. Overexpression of SOX2OT in TAMR cells
increased cell proliferation and cell invasion. However, SOX2OT overexpression did not significantly alter SOX2
levels, suggesting an independent interaction within TAMR cells. Kaplan–Meier plot analysis revealed an inverse
relationship between SOX2OT expression and prognosis in luminal A and B breast cancers. Our findings highlightthe potential role of SOX2OT in TAMR breast cancer progression. The downregulation of SOX2OT in TAMR breast
cancer indicates its involvement in resistance mechanisms. Further studies should explore the intricate interactions
between SOX2OT, SOX2, and TME in breast cancer subtypes.
Keywords Breast cancer, Tamoxifen, Resistance, LncRNA, SOX2OT*Correspondence:
Jin Hyang Jung
jjh01@knu.ac.kr
1
Department of Surgery, School of Medicine, Kyungpook National
University, Daegu, Republic of Korea
2
Cell & Matrix Research Institute, Kyungpook National University, Daegu,
Republic of Korea
3
Department of Oncology/Hematology, School of Medicine, Kyungpook
National University, Daegu, Republic of Korea
4
Department of Pathology, School of Medicine, Kyungpook National
University, Daegu, Republic of Korea
5
Kyungpook National University Chilgok Hospital, Hoguk-ro 807, Buk-gu,
41404 Daegu, Republic of Korea

Introduction
Hormone receptor (HR)-positive breast cancer constitutes a prevalent subtype characterized by the presenceof estrogen receptor (ER) and/or progesterone receptor(PR) positive cells, enabling hormonal stimulation of cancer growth [1, 2]. HR-positive breast cancer accounts forapproximately 60–70% of cases and treatment primarily
relies on hormone therapy, including drugs like tamoxifen and aromatase inhibitors, with targeted therapies
such as CDK4/6 inhibitors sometimes used [3, 4]. HRpositive breast cancer is generally associated with a better prognosis than triple-negative breast cancer (TNBC)
or HER2-positive breast cancer [5–7]. This subtype canbe managed with hormone treatments that either suppress hormones or inhibit HRs [8–10]. However, not allbreast cancer cells may respond consistently, and those© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Lee et al. BMC Molecular and Cell Biology(2024) 25:12

resistant to hormonal treatment can exhibit more aggressive characteristics, making treatment more challenging[11–14].
Long non-coding RNA (lncRNA) is a class of RNA
molecules implicated in cancer development throughvarious mechanisms, which is extensively transcribedin both human and mouse genomes. Characterized as a
non-protein coding transcript longer than 200 nucleotides, lncRNA has been reported to play a significant
role in cancer development and proliferation by acting
as gene expression regulatory factor [15, 16]. They canregulate gene expression by binding to DNA, affecting
epigenetic modifications, and interacting with chromatin-modifying proteins, thereby altering the expression ofgenes involved in cell proliferation, apoptosis, and DNA
repair [17, 18]. LncRNAs can also engage in RNA-protein
interactions, acting as miRNA sponges or influencing
post-transcriptional and post-translational regulation,
resulting in the dysregulation of key oncogenes or tumor
suppressor genes. Additionally, some lncRNAs can contribute to chromosomal instability, influence alternative splicing, and modulate signaling pathways involvedin cancer progression. However, it is crucial to note that
not all lncRNAs have pro-oncogenic roles, as some may
act as tumor suppressors. The specific mechanisms andfunctions of lncRNAs in cancer development can vary
among different cancer types and subtypes, making thema subject of active research with potential therapeutic
implications.
Tamoxifen has been the gold standard treatment for
HR-positive breast cancer for decades [19, 20], andresearchers have been attempting to identify genes or
genomes specifically expressed in tamoxifen-resistantbreast cancer and incorporate them into treatment
mechanisms. Some lncRNAs, including HOX transcript antisense RNA (HOTAIR) and Thymopoietin
antisense transcript 1 (TMPO-AS1), have been shownto contribute to endocrine therapy resistance in breast
cancer through distinct mechanisms [21–24]. One of
the lncRNA, SOX2 has been identified in 43% of basal
cell-like breast cancers [25], and it is significantly associated with CK5/6, EGFR, and vimentin immunoreactivity, while showing an inverse association with estrogenand progesterone receptor status [26]. Although SOX2
mechanism remains largely elusive, several studies haveindicated that SOX2 and SOX2OT are co-expressed atsimilar locations. Furthermore, SOX2OT appears to playa role in SOX2 regulation. However, recent studies havereported that lncRNAs do not simply affect the tumor
cells; instead, cancer promotion, proliferation, and drug
resistance is involved through more complex interactions
with the tumor microenvironment (TME) [27–29].
This study aimed to verify the biologic role of SOX2OT
associated with SOX2 in tamoxifen-resistant breast

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cancer and to understand the potential relationshipbetween TME and lnRNA SOX2OT.

Materials and methods
Cell culture

Luminal type A cell line (MCF-7) was purchased fromthe American Type Culture Collection (ATCC, Manassas, VA, USA). Two different types of tamoxifen-resistant breast cancer cell lines were prepared. One cell line,
MCF-7 Tam1 (CRL-3435TM; TAMR-H), was purchasedfrom ATCC, and the other, a tamoxifen-resistant cellline (TAMR-V), was provided and authenticated by the
University of Virginia Hospital [30, 31]. The TAMR-V
has been continuously exposed to tamoxifen for morethan 10 years to maintain its resistance. MCF-7 cells
were grown in Dulbecco’s Modified Eagle’s Medium(DMEM; Gibco, Grand Island, NY, USA) supplemented
with 10% fetal bovine serum (FBS; Gibco), and TAMR-V
cells were maintained in DMEM containing 10% FBS and10− 7 mol/L TAM (Sigma-Aldrich, St. Louis, MO, USA).
TAMR-H cells were maintained in DMEM containing
10% FBS, 10-µg/mL human insulin (Sigma), and 1-µM
4-hydroxytamoxifen (Sigma).
Next-generation sequencing (NGS) was performed to
distinguish two different types of TAMR cell lines, andthe results are presented as a heatmap (Supplementary
Fig. 1). All cells were cultured for three days to verify
their growth without any treatment, and it was confirmedthat they proliferated well (Supplementary Fig. 2).
Profiling and confirmation of lncRNA expression via
quantitative reverse transcription polymerase chain reaction (RT-qPCR).
Total RNA was isolated from breast cancer cells using
RNAiso Plus (Takara, Otsu, Japan), and its concentration was measured using NanoDrop ND-2000 (Thermo
Scientific, Wilmington, DE, USA). For lncRNA expression profiling, 2-µg total RNA was reverse transcribed to
cDNA using Human LncProfilers™ qPCR Array Kits (System Biosciences, Mountain View, CA, USA).
To confirm the LncProfilers™ qPCR array results, thetotal RNA in breast cancer cells was extracted and random hexamer (ELPIS-Biotech Inc., Daejeon, South
Korea) was applied, following the manufacturer’s protocols. The expression levels of specific lncRNAs, including SOX2 and SOX2OT, were further confirmed by
RT-qPCR. This was performed using both TaqMan Gene
Expression Master Mix and Power SYBR Green PCR
Master Mix (Applied Biosystems, Foster City, CA, USA).
The relative gene expression was normalized to thehousekeeping genes GAPDH and β-actin, employing the 2−ΔΔCT method. Primers and TaqMan probes
used were for SOX2 (5′-AAC CCC AGA TGC ACAACT C-3′, 5′-GCT TAG CCT CGT CGA TGA AC-3′,
Hs04234836-s1), SOX2OT (5′-GCT CGT GGC TTALee et al. BMC Molecular and Cell Biology(2024) 25:12

GGA GAT TG-3′, 5′-CTG GCA AAG CAT GAG GAA
CT-3′, Hs00415716_m1), GAPDH (5′-ACG GGA AGC
TTG TCA AT-3′, 5′-TGG ACT CCA CGA CGT ACT
CA-3′, Hs99999903_m1), and for β-actin (5’-TTG CCG
ACA GGA TGC AGA A-3’ and 5’-GCC GAT CCA CAC
GGA GTA CT-3’). Amplification efficiencies for both
TaqMan and SYBR Green methods were tested, and all
samples were evaluated in triplicate to ensure accuracy
and reproducibility.
Transformation and transfection

SOX2OT sequences were synthesized by Invitrogen(Thermo Fisher Scientific, Waltham, MA, USA), andSOX2OT cDNA fragments were cloned into the pcDNA
3.1 vector to overexpress SOX2OT (Thermo Fisher Scientific). The empty pcDNA3.1 vector was used as thecontrol and pcDNA-SOX2OT or pcDNA-vector transfection into cells was performed using Lipofectamine™
RNAiMAX (Thermo Fisher Scientific), according to the
manufacturer’s protocol.
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium
bromide assay

Cells were inoculated in a six-well plate(4 × 105/well) for the wound healing assay and in a 96-wellplate (5 × 103/well) for the MTT assay. Following culture
overnight, cells were transfected using Lipofectamine™
RNAiMAX reagent (Invitrogen) with the transformed
specific SOX2OT pcDNA3.1(+) plasmid or pcDNA3.1(+)
control plasmid. At 48 h following transfection, the MTT
substrate was prepared in a physiologically balanced
solution, added to cells in culture typically at a final
concentration of 50 µL, and incubated at 37 °C for 4 h.
Following removal of the medium, 150-µL dimethyl sulfoxide was added, and the mixture was shaken for 10 min.
The quantity of formazan (presumably directly proportional to the number of viable cells) was measured byrecording changes in absorbance at 570 nm using microplate spectrophotometer (Agilent Technologies, Winooski, VT, USA). All of the experiments were conductedin triplicate.
Wound healing assay

To determine the effect on cell migration, TAMR-V andTAMR-H cells were seeded in six-well culture plates andtransfected with control pcDNA 3.1(+) or SOX2OTpcDNA 3.1(+). Subsequently, a line was scratched into
the cell monolayer using a sterile pipette tip, and the cells
were further incubated. Images were captured at 0-, 48-,and 72-h timepoints using a microscope and camera system (Olympus Co., Tokyo, Japan). Wound closure wasmeasured using the ImageJ wound healing tool (ImageJ.
Available online: https://imagej.net/Welcome). The data
were representative of three independent experiments.

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Cell invasion assay

For the cell invasion assay, transwell chambers with 8-µm
pores were coated with Matrigel (Corning Inc., Tewksbury, MA, USA) and incubated at 37 °C for 4 h, allowing
it to solidify. Following transfection, cells were resuspended in DMEM containing 1% FBS and plated in theupper chamber at a density of 1 × 104 cells. The lower
chamber contained complete medium supplemented
with 10% FBS. Following 48-h incubation, the cells on theinternal surface of the chamber bottom were wiped witha cotton swab, fixed with 2% paraformaldehyde, stainedwith 0.5% crystal violet, and rinsed with phosphate-buffered saline. Four random fields were selected for each
culture well under a light microscope, and the number ofcells in each view was counted.
Statistical analysis

Data were presented as means (± standard deviation, SD)of three or more independent experiments. The differences in experimental results between the two groups
were analyzed using Student’s t-test, and a statistically significant difference was considered at p < 0.05 or
p < 0.001.

Results
SOX2OT was significantly downregulated in both TAMR
breast cancer cell lines

Using qPCR array analysis, several lncRNAs were evaluated in TAMR-V and TAMR-H breast cancer celllines. Among 90 lncRNAs, we extracted transcriptsthat showed upregulated (> 2-fold) or downregulated(< 0.5-fold) expression, and 24 lncRNAs were analyzed.
While lncRNA anti-NOS2A, EVF1 and EVF2, GAS5family, H19 upstream conserved 1 & 2, HAR1B, andIFG2AS were upregulated, 7SK, NTT, and SOX2OT
were commonly downregulated according to the resultsof each three consecutive tests performed in TAMR-H
and TAMR-V compared with MCF-7 (Fig. 1; Table 1).
Among nine lncRNA candidates, which were consistently
upregulated or downregulated in both TAMR cell lines,
SOX2OT was selected on the basis of literature reviews,
which showed a strong negative association with TAMR
cells [26, 32] (Supplementary Fig. 3).
Expression of SOX2 protein and lncRNA SOX2OT in breast
cancer samples from the Kaplan–Meier (KM) plotter

The genome-wide RNA transcript profile from the KM
plotter (http://kmplot.com) by RNA sequencing (RNAseq) dataset in patients with breast cancer was analyzed and noted direct opposite results with SOX2OTand SOX2 in luminal A and B breast cancers. Although
lncRNA SOX2OT showed a significantly better prognosis with higher SOX2OT expression in luminal A and B
breast cancer (luminal A, HR = 0.30, p = 0.0092; luminal

Lee et al. BMC Molecular and Cell Biology(2024) 25:12

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Fig. 1 Expression profile of various lncRNAs in tamoxifen-resistant breast cancer cell lines (A: TAMR-V; B: TAMR-H) compared to MCF7 cells. While 7SK, NTT,and SOX2OT were downregulated in both TAMR breast cancer cell lines, anti-NOS2A, EVF1 and EVF2, GAS5-family, H19 upstream conserved 1 & 2, HAR1B,
and IGF2AS family were all upregulated in the TAMR cell lines

Table 1 Expression profile of the long non-codingRNAs(lncRNAs) expression levels in tamoxifen-resistant breast
cancer cell lines (TAMR-V and TAMR-H)
Level ofexpression

LncRNA*

Upregulation anti-NOS2A*
EVF1 and
EVF2*
GAS5-family*
H19
upstream
conserved 1& 2*
HAR1B*
IGF2AS(family)*
Downregula- 7SK*
tion
NTT*
SOX2OT*
Control
18 S rRNA
RNU43(snoRNA)GAPDHLAMIN A/C
U6 snRNA*

Expression in
Expression in
TAMR-V/MCF7
TAMR-H/MCF7
Average STDEV Average STDEV
52.82983 16.65489 24.41382 9.56836
34.11834 12.70678 28.66012 10.95019
3.377255 0.835037 4.2247
0.311261
3.027784 1.649675 2.038056 0.510124

9.013918 8.091384 4.250339 4.595892
4.865702 2.040343 1.772124 0.980745
0.656233 0.028282 0.682445 0.061467
0.66975
0.094758
8.822375
1.664141

0.38690.046917
0.791897
0.222279

0.767579
0.044528
11.43136
4.614409

0.308688
0.023905
1.015886
0.336516

0.271698 0.027552 0.983259 0.12848
0.547854 0.02251 0.016867 0.009027
0.31399 0.060132 0.558524 0.121545

These lncRNAs indicated the reference control

B, HR = 0.45, p = 0.0020) (Fig. 2A, B), SOX2 showed conversed results without statistical significance (luminal
A, HR = 1.59, p = 0.0620; luminal B, HR = 1.48, p = 0.1100)(Fig. 2C, D).

TAMR-V and TAMR-H cells were transfected with plasmids
pcDNA3.1 and pcDNA3.1 + SOX2OT

Since the SOX2OT level was decreased in TAMR celllines compared with that in the MCF7 cell in previous
study, SOX2OT expression was intentionally increased to
examine how this reduction affects actual TAMR cancer
cells. The SOX2OT expression was transiently increasedusing SOX2OT transcript introduced pcDNA3.1 plasmid (Fig. 3A), and its transformation was confirmed byagarose gel electrophoresis (Fig. 3B). Both TAMR cells
were transfected with pcDNA3.1 control and pcDNA3.1SOX2OT plasmids, respectively.
The SOX2OT expression level was increased in the
pcDNA 3.1-SOX2OT vector group; however, the SOX2
expression level was unaffected (Fig. 4). These results
indicate that SOX2OT is not directly involved in SOX2
expression in cell lines, and they interacted independently in TAMR cancer cells.
SOX2OT overexpression affects cell proliferation in TAMR
cells

To assess any differences in SOX2OT expression, both
TAMR cell lines were treated with varying doses oftamoxifen and tamoxifen-4OH (control dose [DMSO], 10µM, and 20 µM). To evaluate the sensitivity and changes
in the cell viability of TAMR cells according to the tamoxifen dose, MTT assay was performed. When tamoxifen
was treated to MCF-7, TAMR-V, and TAMR-H breast
cancer cells to confirm tamoxifen resistance, the MCF-7
cell viability was significantly decreased when 20-µM
tamoxifen was treated. However, TAMR-V and TAMRH cells were not decreased. When SOX2OT was overexpressed, the cell viability of TAMR cells were increased,
particularly in TAMR-H cells (p < 0.001) (Fig. 5).

Lee et al. BMC Molecular and Cell Biology(2024) 25:12

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Fig. 2 Kaplan–Meier survival curve based on the expression levels of SOX2 and SOX2OT. (A, B) Overall survival based on the expression levels of SOX2OT
in luminal A and B breast cancer. (C, D) Overall survival based on the expression levels of SOX2 in luminal A and B breast cancer

SOX2OT overexpression tends to promote cell invasion but
not migration in TAMR cells

Overexpression of SOX2OT appeared to enhance
TAMR cell invasion in the Matrigel-coated transwell
assay. Although the difference was not statistically significant, the cells exhibited increased proximity andinvasion following SOX2OT overexpression in TAMR-V

and TAMR-H cell lines (Fig. 6A, B). Cell movement was
tracked for 72 h. No movement was detected in the first
24 h, but a shift in cell movement began at 48 h, becoming more pronounced at 72 h. However, in the woundhealing assay used to assess cell migration, TAMR cells
demonstrated activity levels almost identical to those of
MCF-7 cells (Fig. 6C, D).

Lee et al. BMC Molecular and Cell Biology(2024) 25:12

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Fig. 3 Transfection of pcDNA 3.1 and pcDNA 3.1 with tamoxifen-resistant breast cancer cells. (A) Schematic of the SOX2OT_variant_4_pcDNA 3.1(+) vector. (B) Agarose gel electrophoresis showed SOX2OT variant inserted pcDNA 3.1(+) plasmid

Fig. 4 Expression of SOX2OT and SOX2 in TAMR-V (A) and TAMR-H (B) cells post-transfection. Although SOX2OT expression increased in pcDNA3.1SOX2OT vector group, SOX2 expression was not affected. Comparison of the two groups was performed using Student’s t-test, with error bars representing the standard deviation of at least three independent experiments

Tissue-specific expression of lncRNA SOX2OT in various
breast cancer subtypes

To evaluate the interaction between lncRNAs and tumor
cells through the TME, SOX2OT expression was compared in tissues of different breast cancer subtypes. While
SOX2OT was highly expressed in luminal B type breast

cancer, those in TAMR breast cancers were extremely
suppressed (Fig. 7). Therefore, SOX2OT expression was
confirmed to be consistently downregulated in both
TAMR cell lines as well as TAMR breast cancer tissues.

Lee et al. BMC Molecular and Cell Biology(2024) 25:12

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Fig. 5 Overexpression of SOX2OT affected cell proliferation in tamoxifen-resistant (TAMR) breast cancer cell lines. (A, B) Dose-dependent characterizationof tamoxifen resistance in TAMR-V and TAMR-H breast cancer cells. Parental MCF-7, TAMR breast cancer cells were treated indicated concentrations oftamoxifen or tamoxifen-4OH for 48 h. Viable cells were quantified using MTT assay. (C, D) TAMR cells were overexpressed using SOX2OT plasmid and thentreated with tamoxifen or tamoxifen-4OH. The expression of SOX2OT was observed to be lower in TAMR cells than in control cells, and the cell viability
increased when overexpressed again, especially in TAMR-H cells (p < 0.001). Comparison of the two groups was performed using Student’s t-test, witherror bars representing the standard deviation of at least three independent experiments

Discussion
LncRNAs hold potential as emerging biomarkers andtherapeutic targets for breast cancer [33–35]. Notably,
several lncRNAs have been associated with the acquired
resistance mechanism in HR-positive breast cancer [36,37]. Understanding their biological roles could shed lighton mechanisms to overcome resistance, paving the way
for advancements in therapeutic drug development.
Moreover, SOX2OT has been identified as a novel biomarker linked to tamoxifen-resistant breast cancer, andits association with SOX2 has also been documented[38–40].
In this study, two different types of TAMR breast cancer cell lines were used. One of TAMR cell lines has beendeveloped by treating with real tamoxifen (TAMR-V)
over 10 years, and the other cell line was commerciallyobtained (TAMR-H) by treating with tamoxifen-4OH.
This study utilized multiple cell lines, which, based on
the NGS test, exhibited distinct characteristics. Despite
the variations in TAMR breast cancer cell lines, SOX2OT
consistently showed reduced expression compared to
MCF-7. This indicates a potential relationship between
SOX2OT and the acquisition of TAMR in breast cancer.
The most significant challenge in the experiment was
the sluggish proliferation of HR-positive breast cancer

cells [41]. Actual cell proliferation and migration paces
were very slow compared with those of TNBC or HER2positive breast cancer, making it difficult to determine
the statistical significance in the results. Therefore, developing a novel drug to overcome hormone-resistant breast
cancers is difficult, and HR-positive breast cancers are
considered a refractory disease when they achieve hormone resistance [12, 13, 42].
The TAMR-H cell lines displayed a significant increasein tamoxifen-resistant breast cancer proliferation, while
TAMR-V cell lines showed a pronounced trend. Furthermore, the addition of SOX2OT worsened cell invasion inboth cell lines. In the cell migration test, there was moremigration with SOX2OT even if it did not achieve statistical significance. These findings indicate that lower
SOX2OT levels in hormone-resistant breast cancer areassociated with a better prognosis, similar to HER2-positive or TNBC. Conversely, higher SOX2OT levels correlate with a better prognosis in HR-positive breast cancer,
as seen in the KM plot. Our study indicates that while
SOX2OT overexpression tends to enhance cell invasionin TAMR breast cancer cells, as evidenced by the Matrigel-coated transwell assay, it does not significantly impact
cell migration, as shown in the wound healing assay.
This suggests a complex interaction in which SOX2OTLee et al. BMC Molecular and Cell Biology(2024) 25:12

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Fig. 6 Cell invasion and migration study by SOX2OT overexpression in TAMR breast cancer cell lines. (A, B) Cell invasion by Matrigel-coated transwell
assay showing a tendency of promotion in TAMR cells, which are transfected with SOX2OT overexpression plasmid or control plasmid. Comparison of thetwo groups was performed using Student’s t-test, with error bars representing the standard deviation of at least three independent experiments. (C, D)
Migration of MCF7, TAMR-V and TAMR-H cells transfected with SOX2OT overexpression plasmid or control plasmid are detected by wound healing assay

Lee et al. BMC Molecular and Cell Biology(2024) 25:12

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Fig. 7 Expression of SOX2OT in patients with breast cancer according to the various subtypes of breast cancer. The expression of SOX2OT was highest in
luminal type of breast cancer, and it was lower in TAMR breast cancer than in luminal B type and triple-negative breast cancer

selectively influences the invasive capabilities of cancer
cells without markedly affecting their migratory behavior.
This highlights the nuanced role of lncRNAs in cancer
cell dynamics and underscores the need for further investigation into the specific mechanisms by which SOX2OT
modulates these distinct aspects of cancer progression.
The addition of SOX2OT did not visibly alter SOX2
levels although SOX2OT levels increased significantly
after adding SOX2OT. This suggests a more complex andmultifaceted interaction between SOX2OT and SOX2
in tamoxifen-resistant breast cancer. Previous studies suggest that the diverse interactions between SOX2
and SOX2OT plays a crucial role in the progression ofbreast cancer [32, 43]. Although the proliferation of cells
slowed down, making it challenging to observe statistical significance, our cellular experiments demonstrated aclear association between SOX2OT and the proliferation,
invasion, and migration of tamoxifen-resistant breast
cancer cell lines. Given that these results are consistent
not only in cell lines but also in tissue, it can be inferredthat SOX2OT can promote tumor growth through interactions with the TME rather than directly affecting thecell lines themselves.
This study provides potential insights into the role of
SOX2OT in tamoxifen-resistant breast cancer. We foundthat SOX2OT expression decreases with tamoxifen

resistance, and high levels of SOX2OT enhance the proliferation, invasion, and migration of TAMR cancer cells,
yielding poor outcomes. However, achieving statisticalsignificance was impeded by the slow progression of thedisease. This made it difficult to conclusively determine
the effect of SOX2OT on tamoxifen resistance based
solely on our findings, which was a limitation of thisstudy. A further limitation was the differing results of the
two TAMR cell types. This suggests an unrevealed mechanism at play rather than a simple difference in tamoxifen
type.

Conclusion
In this study, we confirmed that SOX2OT is downregulated in tamoxifen-resistant breast cancer. Moreover, ahigher SOX2OT level is associated with worse outcomes,
including increased TAMR cancer cell proliferation,
invasion, and migration. However, the slow progressionof the disease made it challenging to observe statisticalsignificance. Determining how SOX2OT affects tamoxifen-resistant breast cancer based solely on this study
is difficult. To better understand the impact of lncRNA
SOX2OT on tamoxifen-resistant breast cancer and themechanisms behind this resistance, the relationshipbetween SOX2OT and SOX2 should be further investigated in various breast cancer subtypes.

Lee et al. BMC Molecular and Cell Biology(2024) 25:12

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-024-00510-y.
Supplementary Figure 1: Heatmap of gene expression in TAMR cell lines.
Based on the analysis of 144 genes in the two TAMR cell lines that differed
more than tenfold from MCF7. Red, black, and green represent higher
than average, close to average, and lower than average expressions of aparticular gene, respectively
Supplementary Figure 2: Proliferation of breast cancer cell lines without
treatment. 1.5 × 106 cells from MCF7, TAMR-V, and TAMR-H breast cancer
cell lines were seeded into six wells without treatment, and proliferation
was observed over 72 hours, during which the cells all proliferated wellSupplementary Figure 3: Relative expression levels of SOX2OT and SOX2 invarious breast cancer cell lines

Page 10 of 11

4.
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Acknowledgements
The authors thank the pathologist for facilitating pathological data collection.

12.

Author contributions
Guarantor of the integrity of the study: JL, JHJ; Study concepts: JHJ, JL,
and NJP; Study design: YSC, JL, JHJ, HYP and JYP; Definition of intellectual
content: JL and SJL; Literature research: JHJ, BK, HYP and IHL; Clinical studies:
EAK, JK, HYP, IHL and JL; Data acquisition and analysis: JL, EAK, HYP, IHL andJK; Manuscript preparation: JL, EAK, JK and JHJ; Manuscript editing: JHJ;
Manuscript review: JHJ, YSC; Manuscript revision: JL, EAK and JHJ. All authors
have read and approved the final version of the manuscript.

13.

Funding
This work was supported by Biomedical Research Institute grant, Kyungpook
National University Hospital (2020).

17.

Data availability
The datasets generated and/or analyzed during the current study are available
in the Zenodo repository, https://doi.org/10.5281/zenodo.10869043.

19.

Declarations
Ethics approval and consent to participate
The study protocol was approved by the institutional committee ofKyungpook National University Chilgok Hospital, Daegu, Republic of Korea(KNUCH 2015-05-205), ensuring that it conformed to the ethical guidelinesof the 1975 Declaration of Helsinki. However, because the cell lines cannot
identify the donor, informed consent for each subject was waived.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.

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Received: 31 October 2023 / Accepted: 8 April 2024
27.

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