Normal breast epithelial cells naturally express low levels of the ephrinB2 ligand, while myoepithelial cells express the cognate EphB4 receptor. However, during the proliferative phase of the mammary gland development epithelial cells temporarily express the EphB4 receptor as well (Munarini, Jager et al. 2002). The difficulty in finding a suitable cell line in which to study ephrinB2 biology derives from the fact that many breast epithelial cancer cells acquire the ability to express the EphB4 receptor. In fact, during mammary gland carcinogenesis EphB4 receptor expression is shifted from myoepithelial to epithelial cells (Nikolova, Djonov et al. 1998). Moreover, in most breast cancers the EphB4 receptor is found in 58% of the tumour tissue (Kumar, Singh et al. 2006).
3.1.1
Identifying a suitable epithelial breast cancer cell
model
As a first step, it was necessary to screen a wide range of epithelial breast cancer cell lines to identify cells that don’t express (or express low relative levels of) ephrinB ligands. This was fundamental, as our intention was to create a model system in which we could establish a stable expression of the ephrinB2 protein or ephrinB2 mutants in an epithelial breast cancer cell line.
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We performed Western Blotting (WB) analysis on a panel of 21 breast cancer cell lines to assess the endogenous expression levels of ephrinB ligands. Myoepithelial and HB2 cells (a non-tumorigenic mammary epithelial cell line which displays luminal phenotype) were also included in the screening. WB data shown in figure 3.1 demonstrates that ephrinB ligands are heterogeneously expressed within the panel of cell lines tested. Furthermore, the expression levels vary considerably, with some cell lines expressing high levels of ephrinB ligands (such as MDA-MB-330, CAMA-1, ZR75.1 and SUM149), whilst others expressing smaller amounts (such as HB2, MDA-MB-134, MRSV, BT549, DCIS, GI101 and MDA-MB-231). We have also found that expression of ephrinB ligands was below the level of detection in several cell lines (such as MDA-MB-175, MDA-MB-435, MCF7, T47D, BT474, SKBR3, MYO, BT20, CAL51 and MDA-MB-468). As ephrinB ligands are all relatively similar in size (ephrinB1 = 346 aa; ephrinB2 = 333 aa; ephrinB3 = 340 aa) and are subject to N- glycosylation (data not shown), it was difficult to distinguish between them on the blot when using pan anti-ephrinB pAb. The lower molecular weight band (between 25 and 30 kDa) is probably associated with EphB-induced ligand degradation.
As the preferential receptor for ephrinB2 is the EphB4 receptor (Pasquale 2008), we also analysed endogenous EphB4 levels in these cell lines (figure 3.1). Interestingly, we observed that EphB4 was expressed at different levels in nearly all these cell lines, with only MDA-MB-134, MCF7 and MDA-MB-157 cells expressing minimal amounts or no protein. Within EphB4 expressing cell lines, we noticed that MDA-MB-330, T47D, ZR75.1, BT474, SKBR3, ZR75.30, DCIS, GI101 and MDA-MB-468 expressed higher levels of the EphB4 receptor compared to the remaining cell lines.
One of the main aims of our study was to understand the significance of the ephrinB2 – syntenin-1 axis in the context of epithelial breast cancer. Although syntenin-1 is widely
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expressed in different cell types and usually abundant in epithelial cells (Beekman and Coffer 2008), we analysed endogenous syntenin-1 expression levels in the same panel of cell lines using WB (figure 3.1). As expected, all cell lines expressed medium to high levels of endogenous syntenin-1, apart from MDA-MB-157 cells in which syntenin-1 was barely detectable.
Table 3.1 summarizes the data on expression levels of ephrinB ligands, EphB4 and syntenin-1. Based on these data, the MCF7 cell line (lane 7, figure 3.1) stood out as the best candidate to use as a model system. Although it expresses lower levels of syntenin-1 compared to some of the other cell lines, it presents minimal levels of endogenous EphB4 and no ephrinB ligands.
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Figure 3.1: ephrinB, EphB4 and syntenin-1 expression in a panel of breast cell lines.
A panel of breast cancer cell lines together with myoepithelial and non-tumourigenic HB2 mammary cells was tested for EphB4 receptor, ephrinB ligands and syntenin-1 expression using WB. Cell lysates were provided by Dr Fedor Berditchevski and protein concentrations were measured and standardized to ensure equal loading on SDS-PAGE. Endogenous EphB4 was detected with anti-EphB4 pAb; endogenous ephrinB ligands were detected with anti-ephrinB pAb which recognizes the C-terminal tail of all three ephrinB ligands; endogenous syntenin-1 was detected with anti-syntenin-1 mAb.
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Table 3.1: general indications on the level of overall expression of EphB4, ephrinB ligands and syntenin-1
proteins in 23 epithelial mammary cell lines analyzed via WB analysis. Indications refer to the signal strength detected by WB: none = no signal detected; minimal = signal barely visible; low-high: relative signal strength, with high being the highest signal detected for a specific protein.
Cell line EphB4 expression ephrinB expression syntenin-1 expression WB lane
HB2 LOW MEDIUM MEDIUM 1
MDA-MB-134 MINIMAL MEDIUM LOW 2
MDA-MB-330 MEDIUM HIGH MEDIUM 3
MDA-MB-175 MEDIUM LOW MEDIUM 4
MDA-MB-435 LOW MINIMAL LOW 5
CAMA-1 LOW HIGH MEDIUM 6
MCF7 LOW NONE LOW 7
T47D MEDIUM MINIMAL MEDIUM 8
ZR75.1 HIGH HIGH MEDIUM 9
BT474 HIGH NONE LOW 10
SKBR3 HIGH NONE LOW 11
ZR75.30 MEDIUM LOW MEDIUM 12
MYO LOW LOW LOW 13
MRSV MEDIUM MEDIUM MEDIUM 14
BT20 LOW MINIMAL MEDIUM 15
BT549 LOW MEDIUM LOW 16
CAL51 MEDIUM MINIMAL HIGH 17
DCIS HIGH MEDIUM MEDIUM 18
GI101 HIGH MEDIUM HIGH 19
MDA-MB-157 NONE MEDIUM MINIMAL 20
MDA-MB-231 LOW MEDIUM MEDIUM 21
MDA-MB-468 HIGH MINIMAL HIGH 22