Kits Neodent ®

leukotriene receptors

Our results demonstrate that leukotriene-treatment increases the tumourigenic and metastatic potential of cancer cells likely by expanding the intrinsically more potent subpopulations. Hence, we set out to unravel the molecular mechanisms behind the actions of leukotrienes on the target cancer cells. First of all, we focussed on examining the presence of leukotriene receptors (LTRs) on mammary cancer cells. The Alox5 products leukotrienes signal through their cell surface receptors, LTB4 through BLT1 and BLT2 and cysteinyl-leukotrienes LTC-D-E4 through CysLT1 and CysLT2 and, in fact, leukotriene receptor expression has been previously observed in cancer cells (section 1.2.3.5). We analysed expression of all 4 known leukotriene receptors on the surface of primary MMTV-PyMT, 4T1 and MDA-MB-231 cells by flow cytometry and found BLT2 and CysLT2 to be present on a small proportion. However, we did not detect surface expression of BLT1 or CysLT1 (Fig. 4.7 a-b). The fact that BLT2 and CysLT2 are expressed on small subsets of mammary cancer cells (about 2.5% or 5% respectively) is a very interesting observation in light of metastasis-initiating or cancer stem cell like cells also being small subpopulations of total cancer cells that are expanded upon leukotriene treatment (Fig. 4.6). Hence, we hypothesised that leukotriene receptors might be preferentially present on these cancer cell subpools which intrinsically have a higher tumourigenic or metastatic potential. In fact, we observed a noteworthy expression pattern of both, BLT2 and CysLT2, within the heterogeneous mammary cancer cell population. Leukotriene receptors appear to be highly enriched on the MIC subpopulation of primary MMTV-PyMT cancer cells compared to nonMICs (Fig. 4.7 c-e) as well as on other well-known higher tumourigenic subsets of breast cancer cell lines. These include Aldefluor-active (Ginestier et al., 2007, Hiraga et al., 2011) or CD44-high (Al-Hajj et al., 2003, Sheridan et al., 2006) human MDA-MB- 231 cells and CD49f-high mouse 4T1 cells (Stingl et al., 2006, Yu et al., 2012) (Fig. 4.7 f-h). The enrichment of leukotriene receptor-expressing cells in mammary cancer cell subsets with higher tumour initiation potential suggests that leukotriene receptors themselves might identify a cancer cell population with enhanced

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tumourigenic competence. In order to test this hypothesis, freshly isolated MMTV- PyMT cancer cells were flow-sorted based on the presence (LTR+) or absence (LTR-) of BLT2 and/or CysLT2 followed by two functional tests for tumourigenic potential. Firstly, LTR+ and LTR- MMTV-PyMT cancer cells were cultured in non- adherent conditions to assess their self-renewal ability by quantification of sphere formation. Secondly, these cancer cell subpopulations were orthotopically injected into Rag1-/- mice to compare their tumour formation competence. Strikingly, LTR+ MMTV-PyMT cells showed enhanced sphere formation and mammary tumour initiation ability compared to LTR- cancer cells (Fig. 4.8). Here, we analysed MMTV-PyMT cancer cell populations that contained single BLT2 or CysLT2 as well as double-expressing cells with the reason that both of their ligand types, LTB4 and cysteinyl leukotrienes, enhance tumourigenic and metastatic potential of cancer cells and cause metastasis-initiating and cancer stem cell-like cell expansion (Fig. 4.5 and 4.6). However, the individual potential of BLT2 and CysLT2 single-positive or double-positive mammary cancer cell populations would have to be analysed in dedicated experiments (discussed in detail in section 6.3.2). Nevertheless, in agreement with the enrichment of leukotriene receptor-expressing cells in cancer stem cell-like cell subsets, leukotriene receptor expression identifies a novel subpopulation of MMTV-PyMT cancer cells with enhanced intrinsic tumourigenic potential independent from ligand stimulation.

Next, we aimed to elucidate the signalling downstream of BLT2 and CysLT2 induced by leukotriene stimulation in mammary cancer cells. In line with previous reports on LTB4-mediated signalling (Choi et al., 2010, Kim et al., 2010a, Woo et al., 2002), the analysis of the intracellular response in cancer cells revealed that both, LTB4 and LTC-D-E4, increased levels of intracellular reactive-oxygen-species (ROS) immediately upon stimulation (Fig. 4.9 a). Further, we were intrigued by reports in the literature that demonstrate the LTB4-BLT2 axis-dependent activation of the mitogen-activated protein kinases ERK1/2 in cancer cells, which is directly triggering cell proliferation (Ihara et al., 2007, Park et al., 2012, Tong et al., 2002, Tong et al., 2005, Woo et al., 2002, Zhai et al., 2010). In general, activation of the MAPK/ERK pathway is usually associated with increased cell proliferation and a well-studied target in cancer therapy (section 1.1.3). Hence, the induction of proliferation via ERK1/2 activation in leukotriene receptor-expressing cells would be a likely explanation for the observed leukotriene-mediated enrichment of MICs and

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cancer stem cell-like cells (Fig. 4.6) as these subpopulations are enriched for leukotriene receptor expression (Fig. 4.7). In fact, we observed enhanced activation of ERK1/2 in primary MMTV-PyMT cells starting 30 minutes after stimulation with LTB4 or LTC-D-E4 in adherent culture as determined by Western blotting for phosphorylated ERK1/2 (pERK1/2) on protein lysates (Fig. 4.9 b-d). Moreover, we confirmed that LTB4 treatment leads to activation of ERK1/2 also in the human breast cancer cell line MDA-MB-231 (Fig. 4.9 e). In order to validate that LTR- expressing cells are required within the total cancer cell population to detect a leukotriene-mediated pERK1/2 increase, we largely depleted mammary 4T1 cancer cells from LTR+ cells by flow sorting. Next, we stimulated cancer cells with leukotrienes three days after sorting, at which time the frequency of LTR+ cells was significantly reduced (Fig. 4.10 a). This LTR+ cell-reduced cancer cell population failed to activate ERK1/2 upon LTB4 treatment, while unsorted total 4T1 cancer cells showed a clear increase in ERK1/2 phosphorylation (Fig. 4.10 b-c). In an alternative approach to test the functional relevance of leukotriene receptors for leukotriene-mediated ERK1/2 stimulation, we took advantage of specific leukotriene receptor inhibitors. Both, the BLT2 inhibitor LY255283 and the CysLT2 inhibitor BAY-u9773 interfered with LTB4 or LTC-D-E4-mediated ERK1/2 activation in 4T1 cancer cells, respectively (Fig. 4.11). Of note, leukotrienes and leukotriene receptor inhibitors are supplied in 100% Ethanol, therefore cancer cells are exposed to elevated Ethanol levels in culture when adding both. This high Ethanol concentration (about 2%) led to unexpected reactions of primary MMTV-PyMT cancer cells and made this experiment unfeasible. The 4T1 cancer cell line appeared to be more resistant, however addition of elevated amounts of Ethanol to the culture medium caused an initial decrease of ERK1/2 phosphorylation for 5-15 minutes. Hence, we displayed pERK1/2 levels in 4T1 cancer cells starting at five minutes after leukotriene-stimulation under presence of leukotriene receptor inhibitors. Nevertheless, these data demonstrate that LTB4 and LTC-D-E4 trigger an intracellular increase of ROS levels in concert with ERK1/2 activation in a BLT2 or CysLT2-dependent manner, respectively.

Next, we aimed to determine if this leukotriene-induced ERK1/2 phosphorylation leads to enhanced proliferation of leukotriene receptor-expressing cells, which are enriched among intrinsically highly metastatic or tumourigenic cell subsets. Indeed, three-day leukotriene-treatment of total human MDA-MB-231 cancer cells resulted

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in an increased frequency of LTR+ cells, suggesting that ERK1/2 activation functionally boosts their proliferation (Fig. 4.12 a). Also, leukotriene-stimulation specifically increased the proliferation of MICs, but not nonMICs, in accordance with the MIC subpopulation being enriched for leukotriene receptor-expressing cells (Fig. 4.12 b-d). Cell proliferation was assayed by pulse-chase incorporation of BrdU followed by BrdU detection by flow cytometry. Inhibition of MEK1/2, the upstream kinases of ERK1/2, using the specific inhibitor PD0325901 prevented the leukotriene-mediated elevation in MIC proliferation, stressing its functional dependency on ERK1/2 activation (Fig. 4.12 c).

In summary, the Alox5 products leukotrienes appear to alter the composition of the total mammary cancer cell population by providing a selective proliferative signal and ERK1/2 activation to cancer cell subsets that retain intrinsically higher tumourigenic and metastatic competence due to their enrichment for leukotriene receptor-expressing cells.

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Figure 4-7 Leukotriene receptors BLT2 and CysLT2 are expressed on mouse and human breast cancer cells and enriched in the metastasis-initiating or cancer stem cell-like cell subpopulations

(a-b) Representative flow cytometric analyses of primary MMTV-PyMT cancer cells, the mouse mammary cancer cell line 4T1 and the human breast cancer cell line MDA- MB-231 for expression of the leukotriene B4 receptors 1 (BLT1) and 2 (BLT2) (a) as well as the cysteinyl leukotriene receptors 1 (CysLT1) and 2 (CysLT2) (b). (c-e) Quantification (c-d) and representative flow cytometric analysis (e) of BLT2+ (c) and CysLT2+ cells (d) among MMTV-PyMT non-MICs and MICs (n≥4 per group, 2 independent experiments). (f-h) Flow cytometric quantification of frequencies of leukotriene receptor BLT2+ and CysLT2+ single or double-positive cells (LTR+) among cancer stem cell-like Aldefluor (ALDH)+ or CD44-high subpopulations of human MDA- MB-231 cells (f-g) or CD49f+ mouse 4T1 cells (h), (n≥4 per group, 4 independent experiments). All flow cytometric analyses were gated on alive single cells.

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Figure 4-8 BLT2 and CysLT2 leukotriene receptor expression identifies a novel MMTV-PyMT cancer cell population with enhanced tumourigenic potential

(a-b) Sorted leukotriene receptor (LTR)+ or LTR- MMTV-PyMT tumour cells were plated in non-attachment conditions followed by sphere-quantification at day ten post- seeding for 3 independent experiments (a) or grafted onto the mammary gland of Rag1-/- mice for analysis of tumour formation potential (b). Tumour burden was determined by weighing (n=8 per group pooled from 2 independent experiments isolating cells from different spontaneous primary tumours) after three weeks and (c) representative image of tumours is shown. Sphere formation index (SFI) was calculated as the combination of area of all formed spheres per experiment to incorporate sphere number and size.

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Figure 4-9 LTB4 and cysteinyl leukotriene-stimulation induces ERK1/2 activation in primary MMTV-PyMT cancer cells and human MDA-MB-231 breast cancer cells

(a) Primary MMTV-PyMT cancer cells were stimulated with Ethanol (EtOH) control, LTB4 or LTC-D-E4 in suspension under presence of the DCF-DA dye that becomes fluorescent upon oxidation by reactive oxygen species (ROS) and immediately analysed by flow cytometry. Mean fluorescence intensity relative to unstimulated control is shown over time (4 independent experiments). (b-d) Western blots of ERK1/2 phosphorylation and total ERK1/2 levels (b-c) and quantification (d) of LTB4- or LTC-D- E4-treated MMTV-PyMT cells for the indicated period of time (≥2 independent experiments). Quantification of ERK1/2 phosphorylation in (d) is shown relative to the internal loading control alpha-Vinculin. (e) Dot blot and quantification of ERK1/2 phosphorylation in MDA-MB-231 cells after three-hour stimulation with LTB4 measured by R&D Proteome ProfilerTM Human Phospho-Kinase Array (ARY003B). Quantification of ERK1/2 phosphorylation is relative to reference spot on the array membrane (one membrane array was used).

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Figure 4-10 ERK1/2 phosphorylation upon treatment with LTB4 is dependent on the presence of leukotriene receptor-expressing cells within total mammary 4T1 cancer cells

(a) Flow cytometric quantification of leukotriene receptor expression (BLT2 and CysLT2) of sorted LTR-reduced 4T1 cells, gated on alive single cells (n=3). (b-c) Representative analysis (b) and quantification (c) of 3 independent experiments of Western blots for total ERK1/2 and ERK1/2 phosphorylation relative to internal alpha- Vinculin of unsorted 4T1 cells or 4T1 cells sorted for LTR negativity shown in (a). EtOH: Ethanol.

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Figure 4-11 BLT2 and CysLT2 inhibitors prevent LTB4 and cysteinyl leukotriene- induced ERK1/2 activation in mammary 4T1 cancer cells

(a-d) Analysis and quantification of Western blot for total ERK1/2 and ERK1/2 phosphorylation relative to internal loading control alpha-Vinculin of 4T1 cells following (a-b) LTB4 or (c-d) LTC-D-E4-stimulation for the indicated period of time in presence of BLT2 inhibitor LY255283 or CysLT2 inhibitor BAY-u9773, respectively (n=1). Dotted lines in (b+d) indicate the Ethanol only control level of ERK1/2 phosphorylation. Note the decrease of ERK1/2 phosphorylation observed after 5-15 minutes when adding both, leukotrienes and their receptor inhibitors, is due to the increase in Ethanol concentration. Data are shown as ERK1/2 phosphorylation recovery and increase from 5 to 45 minutes after stimulation. These experiments were performed once.

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Figure 4-12 Leukotriene directly expand the metastasis-initiating MMTV-PyMT cancer cell subpopulation by specifically inducing their proliferation

(a) Flow cytometric quantification of 5 independent experiments of three-day LTB4 and LTC-D-E4-treated MDA-MB-231 cells for frequency of leukotriene receptor (BLT2 and CysLT2)-expressing cells displayed relative to Ethanol (EtOH)-treated control, gated on alive single cells. (b-d) Three-day leukotriene-treated MMTV-PyMT cells in adherent culture were analysed for BrdU incorporation of CD24+CD90+ MICs and CD24+CD90- nonMICs three hours after BrdU addition by flow cytometry, gated on alive single cells. Fold change of percentage of BrdU+ cells among CD24+CD90+ or CD24+CD90- cells is displayed relative to Ethanol (EtOH)-treated control in (b, 3 independent experiments). (c-d) BrdU incorporation upon leukotriene-stimulation in CD24+CD90+ MICs (c) or CD24+CD90- nonMIC MMTV-PyMT cells in additional presence of PD0325901 MEK inhibitor (MEKi, 3 independent experiments). DMSO=Dimethyl sulfoxide-treated control.

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4.2.3 Neutrophil-derived Alox5 metabolites/leukotrienes are required to