Background
Inflammatory breast cancer (IBC), the most aggressive form of breast cancer, represents approximately 2.5% of newly diagnosed breast cancers in the United States [
1]. This percentage reaches an even higher level of 5–10% of breast cancer cases in North African countries such as Tunisia, Morocco, and Egypt [
2,
3]. IBC is a unique disease characterized by erythema, edema of the breast, a “peau d’orange” and formation of lymphatic tumor emboli [
4‐
6]. IBC patients have a poor survival rate with a median of 3 years compared with non-IBC [
1] with no currently available targeted therapies. Based on the surrogate markers estrogen receptor or progesterone receptor (ER/PR) status and human epidermal growth factor receptor (HER)-2 expression, breast cancer can be classified into ER
+ (ER
+/PR
+ and HER-2
−), ER
+HER-2
+ (ER
+/PR
+ and HER-2
+), HER-2
+ (ER
−/PR
− and HER-2
+), and triple negative (ER
−/PR
− and HER-2
−) [
7,
8]. IBC possess the same molecular subtypes as non-IBC [
9,
10], with more than 50% being reported as ER
−, 36–60% HER-2
+, and 30% triple negative according to a multinational IBC registry [
1,
4]. Therefore, the percentage of triple negative breast cancer is higher for IBC compared to non-IBC cases [
7,
11,
12]. Several lines of evidence indicate that the aggressive phenotype of IBC is due to enrichment for chemo- and radioresistant cancer stem cells (CSCs) [
13]. These cells are characterized by self-renewal, unlimited and high proliferative potential, expression of multidrug-resistance proteins, efficient DNA repair capacity and apoptosis resistance [
14,
15]. Using flow cytometry, CSCs can be distinguished from the bulk of the tumor by their expression of cell surface makers CD44 and CD24 (as a CD44
(+)CD24
(-/low) subpopulation) and based on the activity of ALDH1 [
16]. Due to their functional link to therapeutic resistance, CSCs represent an attractive therapeutic target to dampen tumor recurrence [
15,
16].
Syndecan-1 (CD138), a cell surface heparan sulfate proteoglycan, emerges as a candidate target for IBC. It acts as a coreceptor for a multitude of biological factors like growth factors, angiogenic factors, cytokines and chemokines [
17‐
21]. Dysregulated expression and a potential role of Syndecan-1 as a modulator of cell proliferation and invasive growth have been demonstrated in different tumor entities including breast cancer [
22‐
26]. The function and (de)differentiation state of CSCs are substantially modulated by many interconnected signaling pathways e.g. IL-6/STAT3, Hedgehog, WNT and Notch signaling that emerge as relevant therapeutic targets [
27,
28]. Interestingly, we and others uncovered the regulatory role played by Syndecan-1 in IL-6/STAT3 and WNT signaling in the human triple negative (MDA-MB-231) and hormone-receptor positive (MCF-7) non-IBC cell lines [
16], and in Syndecan-1- knockout mice [
29,
30]. While these data suggest that a therapeutic targeting of Syndecan-1 may be a mean of synchronously interfering with multiple relevant pathogenetic routes, the precise role of Syndecan-1 in modulating IBC pathogenesis and its CSC phenotype is still unexplored.
The cell surface epidermal growth factor receptor (EGFR) is overexpressed in approximately 50% of triple negative IBC [
31]. Patients with EGFR-positive tumors are characterized by lower survival rates and are associated with the risk of higher tumor recurrence [
32,
33]. EGFR and/or HER-2 overexpression, and MAPK hyperactivation lead to activation of NFκB associated with ER downregulation in IBC specimens [
34]. Moreover, a significantly positive correlation between EGFR and CD44 expressions exists in breast invasive ductal carcinoma patients and that is associated with the worst prognosis [
35]. Interestingly, in a study of 230 surgical specimens of primary colorectal carcinoma, epithelial positive Syndecan-1 immunostaining was significantly associated with tumor size and EGFR expression [
36].
In this study, we examined the expression of Syndecan-1 and its correlation with the CSC marker CD44, Notch-1 & -3 and EGFR expression in carcinoma tissues of triple negative IBC and non-IBC patients. We further employed siRNA-mediated Syndecan-1 knockdown in the human IBC cell line SUM-149 and HER-2 overexpressing non-IBC SKBR3 cells to decipher its impact on a CSC phenotype (CD44
(+)CD24
(-/low) and ALDH1
+ subpopulations). Of particular importance,, we studied the expression and activity of several distinct signaling pathways relevant for CSC function to address possible underlying molecular mechanism(s) for this effect. Supported by an unbiased cytokine array screening approach, we specifically tested the effect of Syndecan-1 depletion on inflammatory signaling, including the IL-6/STAT3 signaling pathway [
37‐
39]. Furthermore, we investigated a potential impact on the stemness-associated Notch and EGFR pathways [
35,
39]. Our data demonstrate that Syndecan-1 expression is higher in tissues of triple negative IBC than that in non-IBC. Further, Syndecan-1 is a modulator of the CSC phenotype of IBC via IL-6/STAT-3, Notch and EGFR signaling. Therefore, Syndecan-1 may act as a novel marker for this disease and its targeting could have therapeutic implications for IBC patients.
Methods
Antibodies and reagents
The antibodies against p-STAT3(Y705), STAT-3, p-NFκB-p65(Ser276), NFκB-p65, p-Akt(Ser473), Akt and CD44 (clone 156-3-c11) were from Cell Signaling Technology, Inc. (Beverly, MA, USA), gp130 antibody was purchased from R&D Systems (Minneapolis, MN, USA). Anti-human Notch-1 and EGFR antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-human-CD44-FITC, anti-human-CD24-PE, IgG2b-FITC, IgG1-PE antibodies and rhEGF were obtained from Immunotools (Friesoythe, Germany), and anti-Syndecan-1 (clone B-A38) was from Biorad (Hercules, CA, USA). Anti-human-Notch-2-PE & APC, Syndecan-1 (CD138)-PE antibodies were from eBioscience, Inc. (San Diego, CA, USA) and HRP–conjugated secondary antibodies were from KPL (Gaitherburg, MD, USA). Gamma-secretase inhibitor (GSI) was from Calbiochem (Darmstadt, Germany). Media, fetal calf serum (FCS) and tissue culture supplies were from Lonza (Basel, Switzerland). Unless otherwise stated, all chemicals were from Sigma (St. Louis, MO, USA).
Cell culture
The human IBC cell line SUM-149 (a kind gift from Dr. Bonnie Sloane, Wayne State University, Detroit, MI, USA) and the non-IBC cell line SKBR3 (ATCC/LGC Promochem, Wesel, Germany) were maintained in HAM’s-F12 and DMEM containing 10% FCS, 1% glutamine and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 °C, respectively.
Patient’s samples
We enrolled 30 triple negative breast cancer patients from the breast clinic of Ain Shams university hospitals (IBC
n = 13, non-IBC
n = 17). Carcinoma tissues were divided into two parts: one part was fixed in 10% neutral formalin buffered for immunohistochemical staining and the other part was frozen in -80 °C for subsequent isolation of total RNA.
Table 1
Clinical and pathological data of IBC and non-IBC patients
Age |
Range | 29–60 | 35–63 | 0.119a
|
Mean ± SD | 45.15 ± 8.98 | 50.11 ± 9.43 |
NA | 0 | 0 |
Tumor size |
≤ 4 | 2 (16.7%) | 2 (11.8%) | 1.000b
|
> 4 | 10 (83.3%) | 15 (88.2%) |
NA | 1 | 0 |
Lymph node status |
< 4 | 1 (10%) | 7 (41.2%) | 0.098b
|
≥ 4 | 9 (90%) | 10 (58.8%) |
NA | 3 | 0 |
Tumor grade |
G1 | 0 | 0 | 0.332b
|
G2 | 7 (58.3%) | 14 (82.4%) |
G3 | 4 (33.3%) | 3 (17.6%) |
G4 | 1 (8.3%) | 0 |
NA | 1 | 0 |
Lymphovascular invasion |
Negative | 3 (27.3%) | 13 (76.5%) | 0.018*b
|
Positive | 8 (72.7%) | 4 (23.5%) |
NA | 2 | 0 |
Immunohistochemical staining of CD44 and Syndecan-1
Immunohistochemical staining was performed on serial formalin-fixed and paraffin- embedded tissues sectioned at 4 μm-thickness as we previously described [
40]. Tissue sections were deparaffinized by two consecutive incubations in xylene for 10 min each, followed by rehydration through two changes of absolute ethanol, graded decreasing concentrations of ethanol for 5 min each and finally in distilled water. For antigen retrieval, slides were incubated in citrate buffer (pH = 6.0) in a water steamer for 30 min. Slides were left to cool at room temperature for 20 min then washed 3 × 5 min with PBS. Endogenous peroxidase activity of the tissue was blocked with 3% hydrogen peroxide for 5 min (Dual Endogenous Enzyme block, Dako K4065, Glostrup, Denmark) and slides were washed with PBS 3 × 5 min. Tissue sections were blocked in 1% BSA/PBS and incubated overnight at 4 °C in a humidified chamber with the primary anti-CD44 (dilution 1:800) and anti-Syndecan-1 antibodies (dilution 1:100). Afterwards, slides were washed 3 × 5 min and incubated with HRP-Rabbit/Mouse (DAKO EnVision + Dual Link System-HRP (DAB+) for 30 min at room temperature. Then, nuclei were counterstained with hematoxylin, sections were mounted with Permount® and imaged. Negative control slides were run in parallel where primary antibodies were omitted.
siRNA knockdown was performed using a negative control siRNA (negative control #1, Ambion, Cambridgeshire, UK) and siRNA #12634 (Ambion) to target Syndecan-1 coding region. Cancer cell lines were transfected with 20 nM siRNA using Dharmafect reagent (Dharmacon, Lafayette, CO, USA) according to the manufacturer’s instructions. Successful knockdown was confirmed by flow cytometry as previously described [
16,
22].
Flow cytometry
To detect cell surface breast CSC markers, control and Syndecan-1 siRNA transfected cells were incubated with 10 μl of anti-CD44-FITC, anti-CD24-PE and the FITC and PE isotype control antibodies for 30 min at room temperature in the dark. Analogously, cells were analyzed for Syndecan-1 (CD138)-PE in combination with Notch-2-PE or-APC antibodies. Stained cells were analyzed by a cube-8 flow cytometer (Sysmex/Partec, Muenster, Germany). For ALDH1 activity assessment, 1× 106 control and Syndecan-1 siRNA transfected cells were resuspended in assay buffer containing ALDH1 substrate (1 μmol/L). Half of this suspension was incubated with 50 mM ALDH1 inhibitor diethylaminobenzaldehyde (DEAB) as negative control. Afterwards, the cells were incubated for 1 h at 37 °C in water bath in dark with agitation at 10 min interval. Finally, the cells were centrifuged at 400 xg for 5 min and were resuspended in 1 mL assay buffer and stored on ice prior to acquisition by flow cytometry.
Quantitative real-time PCR
Total RNA isolated from cultured cells or frozen tissues using GeneJET RNA Purification Kit (Thermoscientific, Waltham, USA) was reverse transcribed into cDNA using the high capacity cDNA Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was conducted in duplicate for each gene of interest using SYBR Green dye and gene expression levels were measured in a steponeplus detection System (Applied Biosystems). Relative gene expression was evaluated using the 2
-∆∆Ct method after normalization to 18S rRNA or GAPDH as previously described [
22]. Melting curve analysis was performed to confirm specific product amplification. Primers were designed using Primer 3.0 software or referred to the published literature. Primer sequences are listed in Additional file
1: Table S1. For Notch pharmacological inhibition experiments, 1 μM GSI was added for control and Syndecan-1 siRNA transfected SUM-149 cells 24 h before RNA extraction. Data for mRNA expression levels in carcinoma tissues of IBC vs non-IBC (normalized to values of normal tissues collected during reduction mammoplasty) was represented as log2-transformed fold change.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
Briefly, 72 h post transfection, control and Syndecan-1 siRNA transfected cells were washed twice with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitors [
22]. The cell lysates were shaked for 20 min followed by centrifugation at 10,000 x
g for 10 min at 4 °C. Supernatant was collected and protein concentration was determined using Bradford assay (Fermentas, Burlington, ON, Canada). 25–50 μg of protein per lane was separated on 10–12% gels and electrotransferred into polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Immunoblotting was performed using primary antibodies against phospho-NFκB/p65
(Ser276), phospho-STAT3
(Y705), phospho-Akt
(Ser473), Akt, gp130, Notch-1, EGFR and HRP–conjugated secondary antibodies. After washing, specifically bound antibodies were visualized by ECL reaction. Visualized bands were analyzed with ImageJ software (National Institutes of Health, Bethesda, MA, USA) using β-actin or tubulin as loading controls.
Three dimensional (3D) spheroids and colony formation assays
Petri-dishes were coated with 150 μl Cultrex®Basement Membrane Extract (BME) (Trevigen, Inc., MD, USA) and incubated at 37 °C in a CO
2 incubator for 15 min to solidify. Control and Syndecan-1 siRNA transfected SUM-149 and SKBR3 cells were mixed with 2% BME at density of 5 × 10
4 before overlaying onto each coated petridish and incubated for 7–10 days at 37 °C to allow spheroid formation in 3D. The media were changed every 3–4 days, the spheroids were stained with cell tracker red dye, and the number of spheroids (
>50 μm) was counted. To examine the effect of Syndecan-1 silencing on clonogenic ability, 10,000 control and Syndecan-1 knockdown SUM-149 cells were seeded in six-well plates and maintained in Ham-F12 with 10% FBS for 10–14 days as previously performed [
41]. Cells were washed with PBS, fixed in methanol for 20 min and stained with 0.05% crystal violet for 15 min. Excess stain was removed by water and the stain was dissolved in 1 ml 10% glacial acetic acid. The released color was measured by spectrophotometry at 595 nm according to [
42]. Colony formation steps were also performed in presence of 10 ng/mL EGF and 1% FBS (with addition of fresh media at interval 3–4 days) or 1 μM GSI for 24 h followed by exchange with complete growth media.
Secretome profiling of conditioned media of SUM-149 cells grown in 3D spheroids
Cytokines, chemokines and growth factors secreted by control and Syndecan-1-silenced SUM-149 cells grown in 3D were detected in conditioned media (CM) using RayBio cytokine array-C3 (RayBiotech, Inc. GA, USA). All steps needed to form 3D spheroids were analogously performed followed by starvation for 24 h. Media conditioned by the secretome of the cells were collected and subjected to profile 42 biological factors according to the manufacturer’s instructions. The signal intensity of each spot, which represents the secreted chemokine, cytokines, and growth factors was evaluated by subtracting from the background and normalized to positive controls using ImageJ software as we previously described [
40].
Statistical analysis
All Data are presented as mean ± SEM or SD as indicated. Differences among variables were evaluated using χ2, or Fischer’s exact tests. Student’s t-test (for normally distributed data) or Mann-Whitney U-test (for non-normally distributed data) was used for two group comparisons. The statistical difference between more than two groups was evaluated by one-way ANOVA followed by Tukey’s multiple comparison test. The Pearson’s Rank correlation test was used to analyze the correlations. The level of significance was set at p < 0.05. Graphs were plotted and analyses were performed by GraphPad Prism 7 software (San Diego, CA, USA) and IBM SPSS version 22 (Chicago, IL, USA).
Discussion
As Syndecan-1 is an important modulator of inflammation and the CSC phenotype in different experimental models and in cancer [
19,
20], it emerges as a candidate marker for IBC. The current study demonstrates for the first time a higher transcript levels and immunohistochemical staining of Syndecan-1 in clinical samples of triple negative IBC vs non-IBC patients. This is consistent with the prognostic value of Syndecan-1 in different cancer entities, including breast cancer [
55] and in line with the negative correlation between the ER, PR and the proportion of CD138-positive cells in ductal breast carcinoma in situ [
23]. Interestingly, a higher expression of CD44 with a positive correlation with Syndecan-1 exists in tissues of IBC patients. Of note, Syndecan-1 expression is enriched in CD44
+ subpopulation in SUM-149 cells, although this enrichment is less in SKBR3 cells. This is in agreement with the notion of interaction between Syndecan-1 and CD44 promoting glioma cell invasion [
56] and suggesting a physical and functional association as previously described [
57,
58].
To extend our findings to in vitro models and to better understand its functional role, we studied the impact of Syndecan-1 silencing on CSC properties, namely ALDH1 activity and the presence and size of the CD44
(+)CD24
(-/low) subpopulation, in SUM-149 cells. Our data revealed that Syndecan-1 silencing diminished the CD44
(+)CD24
(-/low) and ALDH1-positive subsets compared with controls. These results are consistent with our previous data and other reports demonstrating that Syndecan-1 acts as a regulator of CSCs in triple-negative and ER-positive breast cancer [
16,
29] and in prostate cancer [
43]. These findings were confirmed in SKBR3 cells. ALDH1 positive cells were reduced upon Syndecan-1 silencing in this cell line. Taken together, these data provide evidence for a role of Syndecan-1 as a regulator of a CSC phenotype in different molecular subtypes of IBC and non-IBC cell lines.
One of the characteristic features of CSCs is the ability to form spheroids and colonies [
16,
59]. Our in vitro colony and 3D spheroids formation assays revealed decreased numbers of spheroids formed in 3D and a reduction of colony numbers upon Syndecan-1 knockdown in SUM-149 and SKBR3 cells. This finding is supported by different reports: we have previously shown reduced mammosphere formation and impaired differentiation into cysts in Syndecan-1-depleted MCF-7 cells [
16]. Another study showed that early intervention with a Syndecan-1 inhibitor (OGT2115) or RNAi-mediated Syndecan-1 silencing in a transgenic mouse model of prostate cancer reduced the incidence of adenocarcinoma and the number of c-kit
(+)/CD44
(+) cells in cancer foci [
43].
It is well-known that breast CSCs are substantially regulated by a multitude of signaling pathways, including the IL-6/STAT3, Notch and Hedgehog pathways, and that targeting these pathways represents potential therapeutic approaches [
28]. In this regard, we explored in this study the role of Syndecan-1 in regulating expression of components of the Notch signaling pathway. Interestingly, we found a higher expression of Notch-1 mRNA and a significant positive correlation between Notch-1 & -3 and Syndecan-1 mRNA levels in carcinoma tissues of triple negative IBC vs non-IBC. Moreover, Syndecan-1 is expressed in a Notch-2
(+)-enriched subset with a prominent higher proportion in SUM-149 than that in SKBR3 cells. Additionally, our findings revealed that Syndecan-1 depletion led to downregulation of Notch-1, -3 and -4, and the Notch signaling downstream target Hey-1 at the mRNA levels, and of Notch-1 & -2 at the protein levels in SUM-149 cells. In contrast, only the mRNA level of Notch-3 was reduced in SKBR3 cells upon Syndecan-1 silencing. In support of our data, it was reported that the neural stem cells expressing both Syndecan-1 and Notch-1 have a higher capacity to form neurospheres than singly positive cells [
60]. Another study demonstrated the presence of reciprocal regulation between Notch-2 & -3 and Syndecan-2 in vascular muscle cells with a physical interaction between Syndecan-2 and Notch-3 [
61]. Although Notch-2 has a dual role as a tumor suppressor or oncogene in breast cancer (reviewed in [
62]), a recent study showed that treatment of patient-derived xenografts of epithelial tumors including breast with the Notch-2/Notch-3 antagonist tarextumab suppressed tumor growth and reduced tumor-initiating cell frequency [
39]. In light of this finding, this is the first study reporting that Notch expression is influenced by Syndecan-1 in IBC.
IBC is known to secrete angiogenic and also vasculogenic growth factors, such as VEGF, bFGF, IL-6, and IL-8 [
63]. Coordinate expression and secretion of IL-6, IL-8 and GRO-α via NFκB promote tumorgenesis and are associated with poor outcome in triple negative breast cancer patients [
51]. GRO chemokines are reported to enhance breast cancer metastasis and resistance to chemotherapy [
64]. The maintenance of breast CSCs and their chemoresistance particularly in the basal subtype/triple negative breast cancer is essentially attributed to the synergistic effect between IL-6 [
27,
65] and IL-8 [
66,
67]. Moreover, IL-6 promotes breast cancer bone metastasis through Notch-1 [
68], and induces mammosphere formation in breast cancer cells through Notch-3 [
65]. These data thus integrate the IL-6/STAT3 and Notch signaling pathways with relevance to our findings in IBC. SUM-149 cells secrete detectable levels of IL-6 and IL-8, and their expression enhances mammosphere formation and protects SUM-149 cells from radiation upon treatment with the Notch inhibitor RO4929097 [
41]. We suggest that this effect can be dampened by Syndecan-1 downregulation. Indeed, treatment of SUM-149 cells with Notch inhibitor reduced expression of IL-6, IL-8 and gp130 mRNA levels to the same extent as Syndecan-1 knockdown without any additive effect of Notch inhibitor in Syndecan-1-depleted cells. Strikingly, the same effect was also observed for the direct downstream Notch target gene Hey-1, suggesting that Syndecan-1 and Notch signaling converge on the same downstream target. However, a potential caveat is associated with the interpretation of the gamma-secretase inhibitor study: Pasqualon et al. [
69] have recently shown in a lung cancer model that the transmembrane fragment generated by Syndecan-1 shedding undergoes intramembrane proteolysis by gamma-secretase. If similar mechanisms apply to IBC cells, gamma-secretase inhibitor treatment may not only have directly affected the Notch signaling pathway, but also signaling events triggered by release of the cytoplasmic cleavage fragment of Syndecan-1 [
70]. Overall, our data suggest the existence of a signaling axis involving Syndecan-1, Notch, IL-6/gp130 and IL-8 in IBC. Depletion of Syndecan-1 did not only downregulate expression of IL-6 and IL-8 but also their secretion, thus inhibiting the positive autocrine feedback loop.
There is mounting evidence that the expression of inflammatory cytokines including IL-6 is regulated by the transcription factors NFκB and STAT3 [
71]. In fact, the NFκB transcription factor pathway contributes to the phenotype of IBC and its target genes are elevated in ER- versus ER+ breast tumors [
72]. IL-6 is a direct regulator of breast CSC self-renewal [
65] and IL-6/JAK2/STAT3 pathway is more active in CD44
(+)CD24
(-/low) breast cancer cells compared with other tumor cell types and its inhibition blocks the growth of xenografts [
27]. A constitutively active STAT3 status is found in about 50–60% of breast tumors specifically in IBC after neoadjuvant chemotherapy [
73], which is associated with tumorigenesis and drug resistance [
74]. Moreover, STAT3 inhibition represses CSC traits in HER2-positive breast cancers [
74]. In this context and in agreement with our prior observation in the triple negative MDA-MB-23 l cells [
16], Syndecan-1 knockdown reduced the levels of the activated forms of NFκB and/or STAT3 and downregulated expression of the IL-6/LIF coreceptor gp130 in SUM-149 and SKBR3 cells. Our findings in SKBR3 cells are supported by the observation of an increased IL-6 expression upon HER-2 overexpression, which leads to enhanced breast CSC activity and resistance against anti-HER2 treatment via a STAT3/Akt/NFκB signaling-mediated autocrine-positive feedback loop [
75,
76]. Taken together, this proves the efficacy of Syndecan-1 targeting in dampening the inflammatory signaling mediated by NFκB or STAT3 in the two cellular models of different breast cancer subtypes.
An important cue for IBC pathogenesis and progression is EGFR [
34]. Our data suggest presence of cross-talk between EGFR and Syndecan-1 in IBC. This is reflected by downregulation of EGFR mRNA and protein levels in SUM-149 and the positive correlation in the clinical samples of IBC. Interestingly, we demonstrated that Notch inhibition did not further downregulate expression of EGFR in Syndecan-1-silenced cells, suggesting that Syndecan-1 regulates expression of EGFR via Notch signaling. This is in agreement with the notion of the crosstalk of EGFR with Notch signaling in triple negative breast cancer and their dual inhibition drastically attenuated active Akt
(Ser473 ) [
53,
77]. Given the coreceptor function of Syndecan-1 for growth factors [
18] and downregulation of EGFR expression upon Syndecan-1 silencing, we found downregulation of the EGFR downstream signaling cue pAkt
(Ser473 ) upon treatment with EGF in Syndecan-1 knockdown cells compared to control SUM-149 cells. At the functional level, Syndecan-1 silencing reduced EGF-induced colony formation compared to control SUM-149 cells. Taken together, our results suggest that Syndecan-1 further regulates a CSC phenotype via EGFR expression and implies a role of interconnected Syndecan-1, Notch and EGFR signaling in IBC.
Acknowledgements
The authors would like to thank Prof. Dr. Mohamed Akram Nouh (National Cancer Institute, Cairo University, Egypt) for his help in reading immunohistochemical-staining results, and Birgit Pers and Angelika van Dülmen for technical assistance. Special thanks for Ms. Noura El-Husseiny for her kind assistance in Notch inhibition experiments.