Siglec-8 in Breast Cancer | Encyclopedia MDPI

Siglec-8 in Breast Cancer: Comparison

Please note this is a comparison between Version 1 by Udo Jeschke and Version 4 by Vivi Li.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are involved in various immune cell-mediated diseases. Their role in cancer is poorly investigated, and research focusses on Siglec-expression on immune cells interacting with tumor cells. This study evaluates and provide first evidence for a role of Siglec-8 in breast cancer (BC).

breast cancer
Siglec
Siglec-8
PPARγ
Gal-7
TA-MUC1
prognostic factor
targeted therapy

1. Introduction

Breast cancer (BC) is by far the most frequent malignant tumor in women. In 2018, about 2.1 million women were newly diagnosed with BC, with rising incidence since 1980 ^[1][2].

Breast cancer (BC) is by far the most frequent malignant tumor in women. In 2018, about 2.1 million women were newly diagnosed with BC, with rising incidence since 1980 [1,2].

Sialic acid-binding immunoglobulin-like lectins (short Siglecs) form a group of receptors within the subfamily of I-type (immunoglobulin-type) lectins. I-type lectins, first described in 1995 [3] as integral membrane proteins, often occur with large cytosolic domains and established phosphorylation sites (like Cluster of Differentation molecule 33, 22 (CD33 & CD22) and Myelin Associated Glycoprotein (MAG)). The subfamily of those having the N-terminus consisting of a sialic acid binding lectin domain and whose C-terminal cytoplasmic region typically, but not uniformly, contains conserved signaling domains, was suggested to be called Siglecs in 1998 [4]. Siglecs can be divided into two subgroups: the first is an evolutionary conserved group, consisting of Siglec-1, -2 (CD22), -4 and -15. The second group is formed by Siglec-3 and further CD33 (Siglec-3)-related Siglecs (like Siglec-5 and Siglec-8). These are mainly expressed in various cells of innate immunity (granulocytes, monocytes and macrophages) [5,6]. The various roles of Siglecs in ligand recognition and binding involving cell–cell interactions, but also in intracellular signaling and immune system regulation [6], suggest that they have a major impact on disease pathophysiology, which makes them useful as biomarkers or potential targets.

Sialic acid-binding immunoglobulin-like lectins (short Siglecs) form a group of receptors within the subfamily of I-type (immunoglobulin-type) lectins. I-type lectins, first described in 1995 ^[3] as integral membrane proteins, often occur with large cytosolic domains and established phosphorylation sites (like Cluster of Differentation molecule 33, 22 (CD33 & CD22) and Myelin Associated Glycoprotein (MAG)). The subfamily of those having the N-terminus consisting of a sialic acid binding lectin domain and whose C-terminal cytoplasmic region typically, but not uniformly, contains conserved signaling domains, was suggested to be called Siglecs in 1998 ^[4]. Siglecs can be divided into two subgroups: the first is an evolutionary conserved group, consisting of Siglec-1, -2 (CD22), -4 and -15. The second group is formed by Siglec-3 and further CD33 (Siglec-3)-related Siglecs (like Siglec-5 and Siglec-8). These are mainly expressed in various cells of innate immunity (granulocytes, monocytes and macrophages) ^[5][6]. The various roles of Siglecs in ligand recognition and binding involving cell–cell interactions, but also in intracellular signaling and immune system regulation ^[6], suggest that they have a major impact on disease pathophysiology, which makes them useful as biomarkers or potential targets.

However, only very little is known about the role of Siglecs in general in the development, growth or repression of tumors. First research about Siglecs and tumorigenesis focused on tumor-associated macrophages (TAMs) and the influence of Siglecs in their interaction with tumor cells.

Regarding specifically Siglec-8, it was initially found only on eosinophils, appearing to be the first eosinophil-specific transmembrane receptor [7]. It is now known that Siglec-8 is expressed by eosinophils, mast cells and, in small amounts, by basophils [8]. It is upregulated in the chronically inflamed airway, where it can inhibit inflammation when binding to ligands [9,10]. Recently, many studies have shown the influence of Siglec-8 in eosinophilic disorders [11], especially as a biomarker of eosinophil involvement in allergic and eosinophilic diseases [12]. But Siglec-8 was also detected as a late maturation marker on eosinophils and basophils in patients with chronic eosinophilic leukemia, chronic myelogenous leukemia and on malignant and non-malignant bone marrow mast cells [13]. In eosinophils, interleukin-5 can upregulate Siglec-8 surface expression [14], and Siglec-8 crosslinking with specific antibodies induces eosinophil cell death [15,16]. Interleukin-5 (IL-5) priming enhances the Siglec-8-mediated apoptosis in eosinophils.

Regarding specifically Siglec-8, it was initially found only on eosinophils, appearing to be the first eosinophil-specific transmembrane receptor ^[7]. It is now known that Siglec-8 is expressed by eosinophils, mast cells and, in small amounts, by basophils ^[8]. It is upregulated in the chronically inflamed airway, where it can inhibit inflammation when binding to ligands ^[9][10]. Recently, many studies have shown the influence of Siglec-8 in eosinophilic disorders ^[11], especially as a biomarker of eosinophil involvement in allergic and eosinophilic diseases ^[12]. But Siglec-8 was also detected as a late maturation marker on eosinophils and basophils in patients with chronic eosinophilic leukemia, chronic myelogenous leukemia and on malignant and non-malignant bone marrow mast cells ^[13]. In eosinophils, interleukin-5 can upregulate Siglec-8 surface expression ^[14], and Siglec-8 crosslinking with specific antibodies induces eosinophil cell death ^[15][16]. Interleukin-5 (IL-5) priming enhances the Siglec-8-mediated apoptosis in eosinophils.

Its important role makes Siglec-8 suitable as a target for treatment of eosinophil- and mast cell-related diseases, such as asthma, chronic rhinosinusitis, chronic urticaria, hypereosinophilic syndromes, mast cell and eosinophil malignancies and eosinophilic gastrointestinal disorders [17]. The attention on Siglec-8 as a potential target in many diseases led to the development of a ligand targeting liposomes to cells expressing Siglec-8 [18] and to the establishment of anti-Siglec-8 antibodies. It was shown that anti-Siglec-8 antibodies, in the presence of secondary antibodies, induce apoptosis of eosinophils [19]. Siglec-8 antibodies are currently investigated in mast cell and eosinophilic disorders [20]. In this context, it was also seen that intravenous immunoglobulins contain naturally occurring antibodies against Siglecs. They might be necessary as an immunoregulatory mechanism [21].

Its important role makes Siglec-8 suitable as a target for treatment of eosinophil- and mast cell-related diseases, such as asthma, chronic rhinosinusitis, chronic urticaria, hypereosinophilic syndromes, mast cell and eosinophil malignancies and eosinophilic gastrointestinal disorders ^[17]. The attention on Siglec-8 as a potential target in many diseases led to the development of a ligand targeting liposomes to cells expressing Siglec-8 ^[18] and to the establishment of anti-Siglec-8 antibodies. It was shown that anti-Siglec-8 antibodies, in the presence of secondary antibodies, induce apoptosis of eosinophils ^[19]. Siglec-8 antibodies are currently investigated in mast cell and eosinophilic disorders ^[20]. In this context, it was also seen that intravenous immunoglobulins contain naturally occurring antibodies against Siglecs. They might be necessary as an immunoregulatory mechanism ^[21].

2. Results

2.1. Siglec-8 Expression in Breast Cancer and Correlation to Different Clinical and Pathological Characteristics

Expression of Siglec-8 could be evaluated in 226/235 tissue sections (could not be evaluated in 9 sections due to technical issues). Of these cases, 11 showed no Siglec-8 expression, and the median immune reactivity score (IRS) of Siglec-8 expression was 6. Nuclear staining could not be observed.

The extent of Siglec-8 expression (IRS) correlated significantly with the histopathological subtype (correlation coefficient (CC) −0.18,

= 0009). Kruskal–Wallis test and boxplots analysis showed that Siglec-8 expression was significantly higher in tumors of no special type (NST) compared to non-NST tumors (

= 0.009) (

Figure 1

a).

There was a correlation of borderline significance (CC = 0.152,

= 0.059) between Siglec-8 expression and tumor grading (G). Kruskal–Wallis test showed that Siglec-8 expression was higher in higher tumor grading (G1: median Siglec-8 IRS: 3, G2/3 median Siglec-8 IRS: 6,

= 0.007). Exemplary immunohistochemical Siglec-8-stainings in tumors with different gradings are shown in

Figure 1

. Information about tumor grading is only available in about 70% of all patients, as certain histological subtypes (e.g., lobular, medullar) were not routinely graded at the time these patients were diagnosed with BC.

Figure 1.

Siglec-8 expression dependent on tumor grading. Exemplary immunohistochemical staining results of Siglec-8 in grade 1 (

), 2 (

) and 3 (

c) breast cancer are shown. Magnification: main images ×10, image sections ×25.

) breast cancer are shown. Magnification: main images ×10, image sections ×25.

Spearman analysis revealed that Siglec-8 expression did correlate to ER status (CC = 0.147,

= 0.027) but not to PR status, HER2 amplification or the biological subtype. In the Kruskal–Wallis analysis, the Siglec-8 expression was significantly higher in ER-positive compared to ER-negative tumors (ER-positive: median Siglec-8 IRS 6 vs. in ER-negative: median Siglec-8 IRS 4,

= 0.027), but was not significantly different concerning PR status or HER2 status. The Siglec-8 expression did not differ significantly comparing the different biological subtypes among each other, with partly small sample sizes of single subtypes (Kruskal–Wallis analysis,

= 0.103). However, comparing TNBC to all other subtypes, Siglec-8 expression was significantly lower in TNBC in comparison to all other subtypes (TNBC: median Siglec-8 IRS 4 vs. in the other subtypes: median Siglec-8 IRS 6,

= 0.040).

Siglec-8 expression was also compared to the expression of the tumor-associated epitope of mucin-1 (TA-MUC1, measured by Gatipotuzumab-staining) and to the cytoplasmic levels of Galectin-7 (Gal-7)—both prognostic factors that have already been evaluated in this cohort by our group before ^[22][23]. Siglec-8 expression correlated significantly with TA-MUC1 expression in the cytoplasm (CC = 0.14,

Siglec-8 expression was also compared to the expression of the tumor-associated epitope of mucin-1 (TA-MUC1, measured by Gatipotuzumab-staining) and to the cytoplasmic levels of Galectin-7 (Gal-7)—both prognostic factors that have already been evaluated in this cohort by our group before [22,23]. Siglec-8 expression correlated significantly with TA-MUC1 expression in the cytoplasm (CC = 0.14,

= 0.039, no correlation was found of Siglec-8 with membranous TA-MUC1 expression CC = 0.017,

= 0.803) and to Gal-7 expression (CC = 0.298,

< 0.001).

2.2. Correlation of Siglec-8 Expression with Survival in BC Patients

Median overall survival (OS), progressive-free survival (PFS) and distant disease-free survival (DDFS) was investigated. For survival analyses, tumors were categorized in “Siglec-8 high” and “Siglec-8 low” expressing tumors using receiver operating characteristic (ROC)-curve analysis. An IRS of >3 was considered as high expression of Siglec-8 and IRS of 0–3 as low.

In the overall cohort, Siglec-8 expression (IRS > 3 vs. IRS 0–3) did not correlate with differences in survival regarding PFS (

= 0.971), DDFS (

= 0.941) or OS (

p = 0.850). Due to the correlations of Siglec-8 with Gal-7 and TA-MUC1, survival analyses were also performed regarding these parameters in the context of Siglec-8 expression.

= 0.850). Due to the correlations of Siglec-8 with Gal-7 and TA-MUC1, survival analyses were also performed regarding these parameters in the context of Siglec-8 expression.

Earlier data described that cytoplasmic Gal-7 expression is a prognostic factor for an impaired PFS and DDFS [23]. An IRS > 6 for Gal-7 expression was considered as Gal-7-positive, and an IRS of 0–6 for Gal-7 as negative. When Siglec-8 expression was included in the Gal-7 survival analysis, it was revealed that the prognostic relevance of Gal-7 was only present in the Siglec-8 high-expressing subgroup. In patients with high Siglec-8 expression, high Gal-7 expression was significantly associated with an impaired PFS (

= 0.023,

Figure 2

a). In Siglec-8 low-expressing patients, PFS did not differ significantly between Gal-7 high- and low-expressing patients (

= 0.276). So, patients with both a high Siglec-8 and a high Gal-7 expression showed a significantly impaired PFS compared to all other combinations of high/low either Siglec-8 or Gal-7 expression (median PFS in Gal-7 high and Siglec-8 high 9.76 years, in the others NR,

= 0.032,

Figure 2

c). This subgroup constituted 15.7% of all patients.

Figure 2.

PFS and DDFS in subgroups defined by the combination of Gal-7 and Siglec-8 expression. Kaplan–Meier analyses of PFS (

) and DDFS (

) in subgroups defined by Gal-7 and Siglec-8 expression are shown. In Siglec-8-positive patients, PFS and DDFS differ significantly regarding Gal-7 expression (

), whereas Gal-7 was not significantly associated with PFS and DDFS in Siglec-8-negative patients. Patients in the “Gal-7 high/Siglec-8 high” showed a significantly impaired PFS (

) and DDFS (

d) compared to all other subgroups. PFS = progression-free survival. DDFS = distant disease-free survival. Gal = galectin. IRS = immune reactivity score.

) compared to all other subgroups. PFS = progression-free survival. DDFS = distant disease-free survival. Gal = galectin. IRS = immune reactivity score.

Similar effects could be demonstrated regarding DDFS, where Gal-7 had an association of borderline significance with DDFS only in the Siglec-8 high-expressing subgroup (

= 0.059,

Figure 2

b, not in Siglec-8 low-expressing patients). Patients with both a high Siglec-8 and a high Gal-7 expression also showed a significantly impaired DDFS compared to all other subgroups (median DDFS in all subgroups NR,

= 0.039,

Figure 2

d).

Regarding OS, no differences could be observed for Gal-7, neither in the overall cohort (as already previously described in ^[24]) nor in the subgroups of high and low Siglec-8 expression.

Regarding OS, no differences could be observed for Gal-7, neither in the overall cohort (as already previously described in [39]) nor in the subgroups of high and low Siglec-8 expression.

As described before, membranous TA-MUC1-expression measured by Gatipotuzumab-staining is a prognostic factor for an improved OS, while cytoplasmic TA-MUC1-expression is not [22]. In the current analysis, in addition to the published data, an association of borderline significance of membranous TA-MUC1 expression with an improved DDFS (

= 0.066,

Figure 3

a) could be shown. According to previously published data, an IRS > 2 for membranous TA-MUC1 expression was considered as TA-MUC1-positive, and an IRS of 0–2 for membranous TA-MUC1 as negative.

Figure 3.

Clinical outcome of breast cancer patients regarding TA-MUC1 expression and in subgroups defined by the combination of TA-MUC1 and Siglec-8 expression. Kaplan–Meier analysis of DDFS shows an association of borderline significance between DDFS and TA-MUC1 expression in the overall cohort (

). In patients with low Siglec-8 expression, TA-MUC1 positivity is associated with an improved DDFS (

). However, in patients with high Siglec-8 expression, TA-MUC1 positivity is associated with an improved OS (

c). TA-MUC1 = tumor-associated mucin-1. DDFS = distant disease-free survival. OS = overall survival. IRS = immune reactivity score.

). TA-MUC1 = tumor-associated mucin-1. DDFS = distant disease-free survival. OS = overall survival. IRS = immune reactivity score.

When Siglec-8 expression was included in the TA-MUC1 survival analysis, it could be demonstrated that the prognostic relevance of membranous TA-MUC1 regarding OS was only present in the Siglec-8 high-expressing subgroup: in patients showing a high Siglec-8 expression, a high expression of membranous TA-MUC1 was significantly associated with an improved OS (

= 0.017,

Figure 3

c). In Siglec-8 low-expressing patients however, membranous TA-MUC1 was not significantly associated with OS (

= 0.443).

Siglec-8 furthermore improved the prognostic accuracy of membranous TA-MUC1 regarding DDFS; however, contrary to OS data in the Siglec-8 low-expressing subgroup, in the Siglec-8 low-expressing subgroup, high membranous TA-MUC1 expression was significantly associated with an improved DDFS (

= 0.039,

Figure 3

b). In Siglec-8 high-expressing patients, DDFS did not differ significantly between membranous TA-MUC1-positive and -negative patients (

= 0.307).

Regarding PFS, membranous TA-MUC1 was not a prognostic factor neither in the overall (

= 0.102) nor in the Siglec-8 low- (

= 0.132) or high-expressing cohort (

= 0.205).

2.3. In vitro Experiments with BC Cell Lines

The role of Siglec-8 in BC was further investigated using cell culture models. The expression of Siglec-8 on mRNA level was low in all cell lines investigated. On the protein level, a weak Siglec-8 expression could be detected in MDA-MB231 cells (45 % normalized to β-Actin as a loading control), in MCF7 (24 %) and T47D (20 %) cells.

Due to the correlation of Siglec-8 and Gal-7 expression and their combined prognostic association, a possible influence of Siglec-8 on Gal-7 expression was investigated. When silencing Siglec-8 with three different small interfering RNAs (siRNAs), a significantly downregulated Gal-7 expression in MCF7 cells to 94% (siRNA A,

< 0.001,

= 3), 90% (siRNA B,

< 0.01,

=3) and 86% (siRNA C,

= 0.231,

n = 3) compared to the Siglec-8 wildtype control cells was observed.

= 3) compared to the Siglec-8 wildtype control cells was observed.

As ER status and Siglec-8 expression correlated in the Immunohistochemistry (IHC) analysis, it was examined whether stimulating the ER influences Siglec-8 expression. Stimulating the cells with β-estradiol for 24 or 48 h did not result in any differences in the Siglec-8 expression (data not shown).

Furthermore, literature research revealed a Peroxisome proliferator-activated receptor (PPAR)γ-binding site in the Siglec-8 gene in GeneCards ^[25]. Therefore, the influence of the PPARγ agonist rosiglitazone on Siglec-8 expression on mRNA level was analyzed. Stimulation of MCF7 cells with 1 µg/ml rosiglitazone did not influence Siglec-8 expression. Stimulation with 10 µg/mL rosiglitazone raised the relative Siglec-8 expression up to 171% (

Furthermore, literature research revealed a Peroxisome proliferator-activated receptor (PPAR)γ-binding site in the Siglec-8 gene in GeneCards [24]. Therefore, the influence of the PPARγ agonist rosiglitazone on Siglec-8 expression on mRNA level was analyzed. Stimulation of MCF7 cells with 1 µg/ml rosiglitazone did not influence Siglec-8 expression. Stimulation with 10 µg/mL rosiglitazone raised the relative Siglec-8 expression up to 171% (

= 0.026,

= 3) after 1 h and up to 189% (

< 0.001,

= 3) after 2 h compared to the unstimulated control (normalized to GAPDH as housekeeper gene,

Figure 4

Figure 4.

Mean Siglec-8 expression on mRNA level (calculated with the 2

^−ΔΔCT

method) in MCF7 cells dependent on PPARγ-stimulation. Expression in control cells is displayed and expression in cells stimulated with 1 or 10 µg/mL Rosiglitazone for 1 (

) and 2 (

) hours is normalized on the expression in control cells. GAPDH was used as endogenous control for ΔCT-values and the results are means of triplicates. * indicates a

p-value < 0.05 and ** < 0.01.

-value < 0.05 and ** < 0.01.

3. Discussion

We observed that Siglec-8 is expressed in varying intensity in BC cells without nuclear staining. This expression pattern highlights Siglec-8’s function as a transmembrane protein ^[7]. A comparable staining pattern was observed in eosinophils ^[26]. Interestingly, only two studies used IHC to determine Siglec-8 expression in cancers (in renal and gastric cancer ^[27][28]), with both showing similar expression patterns as in our BC panel.

We observed that Siglec-8 is expressed in varying intensity in BC cells without nuclear staining. This expression pattern highlights Siglec-8’s function as a transmembrane protein [7]. A comparable staining pattern was observed in eosinophils [25]. Interestingly, only two studies used IHC to determine Siglec-8 expression in cancers (in renal and gastric cancer [26,27]), with both showing similar expression patterns as in our BC panel.

We observed higher Siglec-8 expression levels in BCs with a higher grading, which indicates that Siglec-8 expression might be induced when de-differentiation of tumor cells occurs. Siglec-8 expression was significantly higher in ER-positive than in ER-negative tumors and was lowest in TNBC.

Siglec-8 positivity or negativity was not associated with survival rates. However, Siglec-8 expression correlated to the previously identified prognostic factors cytoplasmic Gal-7 levels (negative prognostic factor [23]) and TA-MUC1 expression (membranous expression: positive prognostic factor, cytoplasmatic expression: no prognostic association [22]) in BC.

Evaluating these prognostic factors in the context of Siglec-8 expression, we could demonstrate that a high Gal-7 expression was associated with an impaired PFS in the Siglec-8 high-expressing subgroup. In the Siglec-8 low-expressing subgroup, Gal-7 occurred, but not as a prognostic factor. Furthermore, Siglec-8 knockdown led to a reduced Gal-7 expression, which indicates an interaction of these two proteins. In GeneCards, an interaction of Gal-3—which belongs to the same group as Gal-7—and Siglec-8 is described ^[25]. The interaction of Gal-7 and Siglec-8 might be involved in the mechanism of how Gal-7 levels in tumor cells are regulated: they can be increased by either the induction of mRNA expression or an extracellular to intracellular transfer of Gal-7 ^[29]. In general, it is known that N-acetyllactosamins (LacNAc) epitopes bind to galectins like Gal-1, Gal-3 and Gal-7 ^[30]. For Gal-1, an important role of LacNAcs in the extracellular to intracellular transfer has been shown: extracellular glycans that bear LacNAc epitopes bind Gal-1 and trap it extracellularly. An α-2,6-sialylation of these LacNAc epitopes inhibits the Gal-1 binding and drives the intracellular and then nuclear transfer of Gal-1 ^[31]. A similar mechanism probably exists for Gal-7. Siglec-8 is known to bind sialylated LacNAcs ^[32]. By doing so, it might stabilize the sialylated extracellular LacNAc epitope and promote the liberation of extracellularly bound Gal-7, which could then be transferred intracellularly. The intracellular level of Gal-7 itself can regulate Gal-7 mRNA expression ^[29]. This could be an explanation as to how Siglec-8 knockdown leads to a reduced mRNA expression of Gal-7; however, further functional analyses will have to follow to thoroughly analyze these suggested pathways.

Evaluating these prognostic factors in the context of Siglec-8 expression, we could demonstrate that a high Gal-7 expression was associated with an impaired PFS in the Siglec-8 high-expressing subgroup. In the Siglec-8 low-expressing subgroup, Gal-7 occurred, but not as a prognostic factor. Furthermore, Siglec-8 knockdown led to a reduced Gal-7 expression, which indicates an interaction of these two proteins. In GeneCards, an interaction of Gal-3—which belongs to the same group as Gal-7—and Siglec-8 is described [24]. The interaction of Gal-7 and Siglec-8 might be involved in the mechanism of how Gal-7 levels in tumor cells are regulated: they can be increased by either the induction of mRNA expression or an extracellular to intracellular transfer of Gal-7 [28]. In general, it is known that N-acetyllactosamins (LacNAc) epitopes bind to galectins like Gal-1, Gal-3 and Gal-7 [29]. For Gal-1, an important role of LacNAcs in the extracellular to intracellular transfer has been shown: extracellular glycans that bear LacNAc epitopes bind Gal-1 and trap it extracellularly. An α-2,6-sialylation of these LacNAc epitopes inhibits the Gal-1 binding and drives the intracellular and then nuclear transfer of Gal-1 [30]. A similar mechanism probably exists for Gal-7. Siglec-8 is known to bind sialylated LacNAcs [31]. By doing so, it might stabilize the sialylated extracellular LacNAc epitope and promote the liberation of extracellularly bound Gal-7, which could then be transferred intracellularly. The intracellular level of Gal-7 itself can regulate Gal-7 mRNA expression [28]. This could be an explanation as to how Siglec-8 knockdown leads to a reduced mRNA expression of Gal-7; however, further functional analyses will have to follow to thoroughly analyze these suggested pathways.

On the other hand, the positive prognostic association of membranous TA-MUC1 with OS was also only present in the Siglec-8 high-expressing subgroup. In the Siglec-8 low-expressing subgroup, no associations of TA-MUC1 and OS could be seen. The association between TA-MUC1 and Siglec-8 seems less consistent, as contrary to OS-data, an association of membranous TA-MUC1 expression with DDFS was only present in the Siglec-8 low-expressing subgroup. No data is currently available in the literature about an interaction between Siglec-8 and TA-MUC1. However, when MUC1 is expressed on tumors, it is frequently sialylated ^[21], and Siglecs are known to bind sialic acid structures. A binding of Siglec-9 on macrophages to MUC1 on tumors has been described ^[33]. It might be that Siglec-8 on BC cells co-locates with and therefore “presents” TA-MUC1 or that Siglec-8 binds TA-MUC1 and “transports” it from the cytoplasm to the membrane. Siglec-8 expression correlated to the cytoplasmic TA-MUC1 levels in our study. TA-MUC1 in the cytoplasm is associated with an impaired survival when directly compared to membranous TA-MUC1 ^[22]—Siglec-8 might be involved in a shuttling of TA-MUC1 from cytoplasm to membrane. These hypotheses about an interaction between TA-MUC1 and Siglec-8 could explain why TA-MUC1 does not show a prognostic association regarding OS in the Siglec-8-negative subgroup. A further mechanism could include galectins, as they—including Gal-7—were also found to bind MUC1 ^[34]. However, how the contradictory prognostic effects of cytoplasmatic Gal-7 and membranous TA-MUC1 might be mediated by Siglec-8 needs further research.

On the other hand, the positive prognostic association of membranous TA-MUC1 with OS was also only present in the Siglec-8 high-expressing subgroup. In the Siglec-8 low-expressing subgroup, no associations of TA-MUC1 and OS could be seen. The association between TA-MUC1 and Siglec-8 seems less consistent, as contrary to OS-data, an association of membranous TA-MUC1 expression with DDFS was only present in the Siglec-8 low-expressing subgroup. No data is currently available in the literature about an interaction between Siglec-8 and TA-MUC1. However, when MUC1 is expressed on tumors, it is frequently sialylated [21], and Siglecs are known to bind sialic acid structures. A binding of Siglec-9 on macrophages to MUC1 on tumors has been described [32]. It might be that Siglec-8 on BC cells co-locates with and therefore “presents” TA-MUC1 or that Siglec-8 binds TA-MUC1 and “transports” it from the cytoplasm to the membrane. Siglec-8 expression correlated to the cytoplasmic TA-MUC1 levels in our study. TA-MUC1 in the cytoplasm is associated with an impaired survival when directly compared to membranous TA-MUC1 [22]—Siglec-8 might be involved in a shuttling of TA-MUC1 from cytoplasm to membrane. These hypotheses about an interaction between TA-MUC1 and Siglec-8 could explain why TA-MUC1 does not show a prognostic association regarding OS in the Siglec-8-negative subgroup. A further mechanism could include galectins, as they—including Gal-7—were also found to bind MUC1 [33]. However, how the contradictory prognostic effects of cytoplasmatic Gal-7 and membranous TA-MUC1 might be mediated by Siglec-8 needs further research.

To summarize, survival analyses from our study suggest an association of Siglec-8 with both positive and negative prognostic factors in BC, and a high Siglec-8 expression was especially present in Luminal-like breast cancer. So, inhibiting Siglec-8 in addition to endocrine therapies might be a therapeutic strategy after functional associations have been further clarified. Here, cell culture models can help to study functional effects of Siglec-8.

Although we found a strong Siglec-8 expression in IHC of BC tumors, the Siglec-8 expression on mRNA and protein levels in the BC cells lines we analyzed was quite low. Stimulation with estradiol did not influence Siglec-8 expression. After finding PPARγ binding sites in the transcription factor in the Siglec-8 gene promoter in GeneCards ^[25], we also stimulated BC cell lines with a PPARγ agonist. This led to a stable mRNA elevation of Siglec-8. Siglec-F in mice is assumed to be the equivalent to Siglec-8 in humans. Therefore, experiments in vivo mouse models could be done to verify the effect, even though there are some differences in expression patterns ^[35].

Although we found a strong Siglec-8 expression in IHC of BC tumors, the Siglec-8 expression on mRNA and protein levels in the BC cells lines we analyzed was quite low. Stimulation with estradiol did not influence Siglec-8 expression. After finding PPARγ binding sites in the transcription factor in the Siglec-8 gene promoter in GeneCards [24], we also stimulated BC cell lines with a PPARγ agonist. This led to a stable mRNA elevation of Siglec-8. Siglec-F in mice is assumed to be the equivalent to Siglec-8 in humans. Therefore, experiments in vivo mouse models could be done to verify the effect, even though there are some differences in expression patterns [34].

Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated nuclear hormone receptor that functions as transcription factor and is over-expressed in many tumor types, including BC ^[36][37]. Effects of PPARγ ligands in BC have not been fully understood yet, but data suggest that ligands like Rosiglitazone inhibit proliferation and induce apoptosis ^[38]. Rosiglitazone was investigated in clinical trials ^[39] without achieving breakthroughs, yet ^[24]. The effectiveness of PPARγ therapy could be improved by better understanding the proteins involved in related pathways such as Siglec-8. The numerous effects of anti-PPARγ therapy might include the mechanism by which PPARγ-antagonism reduces Siglec-8 expression.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated nuclear hormone receptor that functions as transcription factor and is over-expressed in many tumor types, including BC [35,36]. Effects of PPARγ ligands in BC have not been fully understood yet, but data suggest that ligands like Rosiglitazone inhibit proliferation and induce apoptosis [37]. Rosiglitazone was investigated in clinical trials [38] without achieving breakthroughs, yet [39]. The effectiveness of PPARγ therapy could be improved by better understanding the proteins involved in related pathways such as Siglec-8. The numerous effects of anti-PPARγ therapy might include the mechanism by which PPARγ-antagonism reduces Siglec-8 expression.

The role of Siglecs in tumors, including possible therapeutic targeting, is currently being investigated. This research focuses on the role of Siglecs as targetable immune checkpoints ^[40]. However, all these studies aim to target Siglecs on immune cells in the tumor microenvironment. In contrast, our study focused on the role of Siglec-8 on the tumor itself. Regarding the role of Siglec-8 on tumors, Siglec-8 expression (measured by IHC) was identified as a potential independent prognostic biomarker of clear cell renal cell carcinoma, where a high expression correlated with an impaired OS and DDFS ^[27]. In contrast, low Siglec-8 expression (IHC) was an independent poor prognosticator for OS in patients with gastric cancer after surgical resection. This was especially seen in higher TNM stages, and the authors suggested that low Siglec-8 expression could be used as a marker to identify patients needing more aggressive adjuvant therapies ^[28].

The role of Siglecs in tumors, including possible therapeutic targeting, is currently being investigated. This research focuses on the role of Siglecs as targetable immune checkpoints [40]. However, all these studies aim to target Siglecs on immune cells in the tumor microenvironment. In contrast, our study focused on the role of Siglec-8 on the tumor itself. Regarding the role of Siglec-8 on tumors, Siglec-8 expression (measured by IHC) was identified as a potential independent prognostic biomarker of clear cell renal cell carcinoma, where a high expression correlated with an impaired OS and DDFS [26]. In contrast, low Siglec-8 expression (IHC) was an independent poor prognosticator for OS in patients with gastric cancer after surgical resection. This was especially seen in higher TNM stages, and the authors suggested that low Siglec-8 expression could be used as a marker to identify patients needing more aggressive adjuvant therapies [27].

Interestingly, already, in 2000, an anti-CD33 (= Siglec-3) antibody as part of an antibody–drug conjugate was approved for treating acute myeloid leukemia [41]. After the role of Siglec-8 has been fully clarified in BC, using it in an antibody–drug conjugate could be an option.

Our study gives first evidence about a role of Siglec-8 expression in BC. Further studies will have to clarify functional aspects to evaluate its role as a possible therapeutic target.

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