17β-Estradiol Modulates the Expression of CD44 and CD326 in Estrogen-Sensitive Breast Cancer Cells

With the emergence of Molecular Targeted Therapy, the interest in studying immunogenetic components that act in carcinogenesis has grown. The role of the estrogen receptor (ER) in initiation and progression of breast cancer is well documented and the estrogen treatment may affect expression of proteins described as tumor stem cell biomarkers in estrogen-sensitive breast cancer. The aim of this study is to analyze the expression of CD44 and CD326 on MCF-7 (ER+) and MDA-MB-231 (ER-) cell lines treated with 17β-estradiol for different periods. Our results indicate that 17β-estradiol can modulate CD44 and CD326 expression in breast cancer cells that have functional estrogen receptors in a time dependent manner. To our knowledge, this is the rst study to investigate the inuence of 17β-estradiol on CD44 and CD326 expression in MCF-7 and MDA-MB-231 cell lines. Further investigations with primary patient samples and their cultures will enhance our knowledge on the effect of hormones on breast cancer.


Introduction
Breast cancer (BC) is the most prevalent tumor among women, accounting for 1 in 4 female cancers, and representing 6.6% of deaths from malignant tumors [1]. BC is a heterogeneous disease and its treatment depends on a molecular classi cation based on the presence of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2) [2]. ER-positive BC further divides into Luminal A and Luminal B subtypes [3]. Generally, well-differentiated tumors with a relatively better prognosis are known to be ER-positive, which account for approximately 60-65% of primary breast cancers [4]. In contrast, triple-negative tumors that lack of ER, PR and HER2 expression account for 15% of all breast cancers are reported to be associated with higher mortality [5]. In a particular way, ER plays a key role in mediating endogenous hormones and therapeutic agents: upon ligand binding, ER modulates various genes' expression either directly by interacting with DNA or indirectly via other transcription factors [6]. There is also evidence that estrogen is capable of inducing BC cell proliferation and cell cycle progression by inhibiting proteins that control cell cycle [7].
Cell lines are valuable research tools for evaluating both the genetics and potential treatment options for BC [8]. Belonging to the luminal A molecular subtype, ER-positive and PR-positive MCF-7 cell line is a noninvasive cell line with low metastatic potential [9]. Being ER-positive cells, the MCF7 cell line depend on estrogen to proliferate. While ER expression is reported to be relatively weak in the parental line compared to tamoxifen-resistant sub-lines, parental MCF7 cells still express 17β-estradiol receptor [9]. On the other hand, poorly differentiated and invasive MDA-MB-231 cells have high metastatic potential and represent the triple negative BC phenotype [8].
With the discovery of cancer stem cells, studying cellular subpopulations expressing certain membrane proteins which are shown to be linked with breast cancer growth has gained importance [10,11]. This hypothesis, which was rst suggested for initiation of acute myeloid leukemia, is also applied to breast cancer [12]. In this context, CD44 + /CD24 low/− cells in BC have shown to exert stem-cell like properties such as high proliferation potential and ability of differentiating while their growth rate decrease once they are differentiated into other well differentiated cell types [13]. BC tumors are heterogeneous with both highly tumorigenic and non-tumorigenic populations, and it is crucial to target tumorigenic cells as these would lead to tumor relapse if they are not eradicated with an effective treatment [13]. Altered expression of anti-apoptotic proteins and certain membrane transporters or multiple drug resistance observed in normal stem cells may also be applied to this highly tumorigenic cell population observed in BC, suggesting targeting this population would be more e cient in cancer therapy [13].
CD44, a non-kinase transmembrane proteoglycan, is a multifunctional cell surface adhesion receptor that is widely expressed on various tissues as well as cancer cells and is a well-known marker for cancer stem cells [14]. CD44 binds to the extracellular matrix component hyaluronic acid, which is expressed by both stromal and cancer cells to initiate various signal pathways that leads to proliferation, migration and invasion [15]. Three CD44 variants, CD44v3, CD44v5, and CD44v6 have shown to be linked with BC metastases [16]. In addition, CD44 positivity, either alone or in combination with other stem cell markers is reported to be associated with tumorigenic potential [16].
Epithelial cell adhesion molecule (EpCAM, also CD326) is a transmembrane glycoprotein which is also involved in cellular signaling, proliferation, migration and differentiation [17]. CD326 is a well-established epithelial cell marker which is one of the most common expressed tumor-associated antigens found in various cancers including BC [18]. CD326 is involved in cell adhesion in a manner that is independent of Ca2 + and also regulates other adhesion molecules' functions [19]. CD326-expressing cells show low contact inhibition and polarization [19]. The in uence of CD326 expression on prognosis depends on the type of tumor, and a correlation with poor prognosis in BC has been reported [20]. Currently, two available approaches for CD326 expressing tumor cells are immunotherapy and targeted drug delivery [19].
In this study, we hypothesized that estrogen treatment may affect expression of proteins described as tumor stem cell biomarkers in estrogen-sensitive BC. Therefore, the aim of this study is to analyze protein Cell culture MCF-7 (passage 24) and MDA-MB-231 (passage 39) cell lines were cultured in high glucose DMEM supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin antibiotic solution. Cells were seeded in a 6-well culture plate as 1x10 5 cells per well and incubated overnight to allow cell attachment in a humidi ed environment at 37°C and 5% CO 2 . 17β-estradiol was dissolved in DMSO to obtain 5 mM stock solution. This solution was further diluted with medium to prepare complete cell culture media supplemented with 100 nM 17β-estradiol. Cells cultured without hormone addition were used as control. Protein expressions were evaluated on 6, 24, 48 and 72 hours of culture by ow cytometry.
Measurement of protein expression using ow cytometry Cells were detached with Trypsin-EDTA solution at respective timepoints and washed twice with DPBS, followed with staining with mouse anti-human CD44-PE and mouse anti-human CD326-PerCP/eFluor710 antibodies by incubating at room temperature for 15 minutes in the dark. Analyses were performed with Navios ow cytometry system (Beckman Coulter, USA). Cells were gated according to Side Scatter/Forward Scatter Signals. Each analysis was performed as triplicates and 25x10 3 events per tube were analyzed. Data acquisition was performed using CXP software.

Statistical analysis
Statistical analysis was performed using GraphPad Prism software (version 8). Two-way ANOVA, followed by Tukey's multiple comparison tests was performed considering two independent variables: treatment (control and treated with 17β-estradiol) and time (6h, 24h, 48h and 72h). p values lower than 0.05 were considered as statistically signi cant. Figure 1 shows representative dot plots indicating CD44 and CD326 protein expressions on cells in a time dependent manner. The main ndings are described below.

Results
Expression of CD44 and CD326 on MCF-7 cell line 17β-estradiol treatment signi cantly reduced CD44 expression compared to control group on 6h (p < 0.001). However, no difference between cells analyzed on 24, 48 and 72h was observed. Yet, CD44 protein levels on 6h was found signi cantly lower compared to 24h (p < 0.01) and 72h (p < 0.001) (Fig. 2.a). CD326 were found to be expressed at different levels on untreated MCF-7 cells in a time dependent manner: Its expression was slightly decreased on 24h compared to 6 h (p < 0.05), and later signi cantly increased on 48h (p < 0.0001). On 72h, CD326 was decreased again compared to 48h (p < 0.001), which may indicate that CD326 expression dynamically changes in cell culture over time. When treated with 17β-estradiol, CD326 expression decreased slightly but not signi cantly within the rst 24h. On 48h, 17βestradiol signi cantly decreased CD326 expression compared to control (p < 0.05). Moreover, incubation with 17β-estradiol up to 72h further decreased CD326 compared to 48h (p < 0.01), yet no signi cant difference between control at respective timepoint was detected (Fig. 2.c).
Ratios of CD44 + CD326 + double positive cells were altered in a time dependent manner; slightly decreasing on 24h compared to 6h (p < 0.05), increasing signi cantly in 48h compared to 24h (p < 0.001) and further decreasing on 72h compared to 48h (p < 0.01). When comparing 17β-estradiol treated cells with their untreated counterparts, signi cant decreases in terms of double positive cells were detected on 6h (p < 0.05) and 48h (p < 0.05). Treatment decreased CD44-CD326-cells. In addition, there was a decrease in double-negative cells in the 72h incubation compared to 48h (p < 0.05) (Fig. 2.e). On the other hand, treatment with 17β-estradiol only signi cantly increased the double-negative population compared to the control group on 6h (p < 0.001). In comparison with 6h, treatment with 17β-estradiol signi cantly decreased double-negative cells on 24h (p < 0.05), 48h (p < 0.05) and 72h (p < 0.001), although it was not observed difference between treatments and their respective controls ( Fig. 2.g) Altogether, our data shows that 17β-estradiol exerts its effects on MCF-7 cells' CD44 expression within rst 6h and loses its effects by 24h. On the contrary, 17β-estradiol does not affect CD326 expression until 48h, which may indicate that CD44 and CD326 expressions are controlled by distinct molecular mechanisms.

Discussion
With the emergence of molecular targeted therapy, the interest in studying the immunogenetic components that act in carcinogenesis has increased [21]. Hyaluronic acid receptor CD44 regulates cellextracellular matrix and cell-cell interactions and is a well-known breast cancer stem cell (BCSC) marker [22]. CD44 can also upregulate regulate immune checkpoint protein Programmed death-ligand 1 expression to prevent tumor from eradication by the immune system [23]. BCSCs can exist in both epithelial-like and mesenchymal-like states re ecting their healthy counterparts and under the regulation of tumor microenvironment, they undergo reversible epithelial-to-mesenchymal and mesenchymal-toepithelial transitions [24]. Moreover, BCSCs can modulate hyaluronic acid protein levels for promoting migration of tumor-associated macrophages in the CSC niches to maintain the proliferative capacity [25]. Epithelial adhesion molecule, CD326 is normally reside in the basolateral membrane while it translocates to the outer lea et of the cell membrane during cancer progression [26]. Overexpression of CD326, one of the rst biomarkers of epithelial cancers discovered in 1970s on the cell membrane [19], is detected in most of human epithelial carcinomas including BC, and attracted researchers' attention as a target for immunotherapy [27]. Moreover, as a signaling receptor modulating stem cell plasticity and involving in regulation of malignant transformation, CD326 is considered as a marker of tumor initiating cells [28]. In fact, Braun et al. revealed that administration of murine monoclonal antibody 17-1A (Edrecolomab) can contribute to disease-free survival in breast cancer [29].
The role of estrogen on initiation and progression of breast cancer is well documented in the literature [2,6]. Here, we aimed to reveal if 17β-estradiol has a modulatory effect on CD44 and CD326 protein expression levels in MCF-7 (ER+) and MDA-MB-231 (ER-) cell lines in a time dependent manner. Our results indicate that 17β-estradiol may modulate CD44 and CD326 expression in ER + breast cancer cells, as well as having no signi cant effect on ER-cells.
CD44 protein expression in MCF-7 cells is previously shown to be associated with higher resistance to hormonal treatments and higher invasive capacity [30]. In a murine breast cancer xenograft model, CD44 targeting with a monoclonal antibody, P245, is shown to inhibit tumor growth, decrease chemotherapy resistance in addition to preventing reoccurrence [31]. Moreover, CD44 may contribute to the silencing of genes in uenced by estrogen treatment through mechanisms that are independent of the ER [32]. In our experiments, the MCF-7 control group showed a high percentage of CD44 + cells, which is consistent with previous data [33]. 17β-estradiol treatment led to a small yet signi cant reduction in CD44 percentages.
Thus, these data may suggest that estrogen treatment may decrease CD44 protein expression on ER + breast cancer cells to increase treatment effectivity.
It is well established that MCF-7 cell line has prominent CD326 expression, which has motivated further studies using this protein as a possible target for anticancer therapies [34,35]. Our data indicates that untreated MCF-7 cells' CD326 expression levels are compatible with the literature ndings [36]. In addition, 17β-estradiol treatment reduced CD326 protein levels signi cantly after 48h. Thus, it is possible that the signaling pathways triggered by the estrogen receptor in this cell line act by modulating CD326 expression.
Increased CD44 expression in MDA-MB-231 cells has been associated with elevated metastatic potential [13,37]. In addition, recent studies have shown CD44 as a potential target for disabling immunosuppression mechanisms in triple-negative cancer cells [23]. In our experiments, both the 17βestradiol treated and control groups showed high CD44 protein levels, which is consistent with previous data [33]. Since triple negative breast cancer cells lack estrogen receptors, 17β-estradiol treatment alone may not be enough to alter CD44 expression when compared to the control. However, alteration of CD44 expression on control group may be associated with time rather than 17β-estradiol treatment.
In cell lines of mesenchymal origin such as MDA-MB-231, CD326 expression is low and tumor cells grow independently of the signaling of this membrane protein [38]. Previously, MDA-MB-231 cells that were genetically engineered to overexpress CD326 were used in a study that found decreased migration and tumor invasion in an animal model, as well as increased in ammation and innate immune responses [38]. In our experiment, MDA-MB-231 cells had CD326 expression which is consistent with previous studies [36,39]. 17β-estradiol had no effect on CD326 expression, which was expected, since MDA-MB-231 cells lack ER. Therefore, even in the perspective of genetically engineered cells to overexpress CD326, 17β-estradiol is likely to have no in uence on the expression of this membrane protein.
In the context of cancer stem cell development, the importance of CD44 and CD326 as biomarkers of cell subpopulations responsible for sustaining tumor growth is evident [10]. In fact, molecular therapeutic strategies targeting cells expressing these proteins may be the pathway to successful treatments [40]. Our ndings con rm that CD44 and CD326 are relevantly expressed in certain breast cancer cell types and demonstrate that the expression of these biomarkers may be in uenced by the hormonal microenvironment.
In conclusion, our results indicate that 17β-estradiol can modulate CD44 and CD326 expression in breast cancer cells that have functional estrogen receptors in a time dependent manner. To our knowledge, this is the rst study to investigate the in uence of 17β-estradiol on CD44 and CD326 expression in MCF-7 and MDA-MB-231 cell lines. Further investigations with primary patient samples and their cultures will enhance our knowledge on the effect of hormones on breast cancer.
was written by DPMB and ANA. BA and GYD commented on previous versions of the manuscript. All authors read and approved the nal manuscript.