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. 2014 Oct 15;5(19):9322-34.
doi: 10.18632/oncotarget.2427.

P2Y2R activation by nucleotides released from the highly metastatic breast cancer cell MDA-MB-231 contributes to pre-metastatic niche formation by mediating lysyl oxidase secretion, collagen crosslinking, and monocyte recruitment

Young Nak Joo  1 , Hana Jin  1 , So Young Eun  1 , Sang Won Park  1 , Ki Churl Chang  1 , Hye Jung Kim  1
Affiliations

P2Y2R activation by nucleotides released from the highly metastatic breast cancer cell MDA-MB-231 contributes to pre-metastatic niche formation by mediating lysyl oxidase secretion, collagen crosslinking, and monocyte recruitment

Young Nak Joo et al. Oncotarget. .

Abstract

Tumor microenvironmental hypoxia induces hypoxia inducible factor-1α (HIF-1α) overexpression, leading to the release of lysyl oxidase (LOX), which crosslinks collagen at distant sites to facilitate environmental changes that allow cancer cells to easily metastasize. Our previous study showed that activation of the P2Y2 receptor (P2Y2R) by ATP released from MDA-MB-231 cells increased MDA-MB-231 cell invasion through endothelial cells. Therefore, in this study, we investigated the role of P2Y2R in breast cancer cell metastasis to distant sites. ATP or UTP released from hypoxia-treated MDA-MB-231 cells induced HIF-1α expression and LOX secretion by the activation of P2Y2R, and this phenomenon was significantly reduced in P2Y2R-depleted MDA-MB-231 cells. Furthermore, P2Y2R-mediated LOX release induced collagen crosslinking in an in vitro model. Finally, nude mice injected with MDA-MB-231 cells showed high levels of LOX secretion, crosslinked collagen and CD11b+ BMDC recruitment in the lung; however, mice that were injected with P2Y2R-depleted MDA-MB-231 cells did not exhibit these changes. These results demonstrate that P2Y2R plays an important role in activation of the HIF-1α-LOX axis, the induction of collagen crosslinking and the recruitment of CD11b+ BMDCs. Furthermore, P2Y2R activation by nucleotides recruits THP-1 monocytes, resulting in primary tumor progression and pre-metastatic niche formation.

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Conflict of interest statement

Conflict of interest

Authors declare no conflicts of interest.

Figures

Fig.1
Fig.1. Hypoxia and ATP/UTP induced HIF-1α expression through the activation of P2Y2R in MDA-MB-231 but not MCF-7 cells
(A) MDA-MB-231 and MCF-7 cells were exposed to hypoxia (2% O2) for various time periods (1 - 24 h), and HIF-1α expression was determined from cell lysates by Western blotting as described in the Methods. (B) MDA-MB-231 cells were pretreated with or without apyrase for 1 h and then exposed to hypoxia for 8 h. HIF-1α expression levels were determined as described above. Data represent mean values ± SEM of three independent experiments (compared with the control, **P < 0.01; compared with hypoxia, ††P < 0.01). (C, D) MDA-MB-231 and MCF-7 cells were treated with ATP (10 μM) or UTP (10 μM) in a time-dependent manner (1 - 24 h), and HIF-1α expression levels were determined as above. (E) Control siRNA- or P2Y2R siRNA-transfected MDA-MB-231 cells were treated with ATP or UTP (10 μM) for 8 h, and HIF-1α protein levels were determined by Western blot analysis. Data represent mean values ± SEM of three independent experiments (compared with control, **P < 0.01, *P < 0.05).
Fig.2
Fig.2. P2Y2R activation by ATP or UTP induced LOX secretion in MDA-MB-231 cells but not in MCF-7 cells
(A) MDA-MB-231 and MCF-7 cells were exposed to hypoxia for 4, 8, and 16 h. After incubation for the indicated time, CM were collected and concentrated 20 times, and LOX levels in the CM were determined by Western blot analysis as described in the Methods. (B) MDA-MB-231 cells were pretreated with or without apyrase for 1 h and then exposed to hypoxia for 8 h. LOX levels from the CM were determined as described above. (C, D) MDA-MB-231 and MCF-7 cells were treated with ATP (10 μM) or UTP (10 μM) for 4, 8, and 16 h, and then LOX levels in the CM were determined by Western blotting as described above. (E) Control siRNA- or P2Y2R siRNA-transfected MDA-MB-231 cells were treated with ATP (10 μM) or UTP (10 μM) for 8 h, and LOX levels in the CM were determined as described above. Data represent mean values ± SEM of three independent experiments (compared with control, **P < 0.01, *P < 0.05).
Fig.3
Fig.3. P2Y2R-mediated LOX release induced collagen crosslinking
CTRL siRNA-transfected MDA-MB-231 cells (A) or P2Y2R siRNA-transfected MDA-MB-231 cells (B) were pretreated with βAPN (300 μM), a LOX inhibitor, for 1 h and then stimulated with ATP (10 μM) or UTP (10 μM) for 8 h. CM from the cells were concentrated 20-fold, and 100 μl of CM was mixed with collagen type I solution (4 mg/ml, 100 μl) as described in the Methods. After a 16-h incubation in a 37°C cell culture incubator, the amount of crosslinked collagen was determined using a fluorescence microscope. Data represent mean values ± SEM of three independent experiments in triplicate (compared with the control, **P < 0.01; compared with ATP or UTP treatment, ††P < 0.01).
Fig.4
Fig.4. P2Y2R expression was increased in THP-1 human monocytes in hypoxia, and P2Y2R activation by ATP or UTP induced THP-1 migration and the release of MMPs
A) THP-1 human monocytes were incubated in hypoxia for 24 h, and then P2Y2R mRNA was analyzed by RT-PCR. Results were confirmed by repeated experiments. B) THP-1 cells were transfected with control siRNA or P2Y2R siRNA (100 nM), and the efficiency of siRNA transfection was confirmed by RT-PCR. C-E) Control siRNA- or P2Y2R siRNA-transfected THP-1 cells (2 × 105 cells) were added to 24-well cell culture inserts, and supernatants from MDA-MB-231 cells exposed to hypoxia in the presence or absence of apyrase (5 U/ml), an enzyme that rapidly hydrolyzes extracellular nucleotide 5′-diphosphates and triphosphates, for 8 h (D) or media containing ATP (10 μM) or UTP (10 μM) (E) were added to the lower chamber of the Transwell. After incubation for 6 h in a 37°C cell culture incubator, the cells that had migrated through the insert membranes were stained with DAPI and counted under a fluorescence microscope. Values represent the means ± SEM of 3 independent experiments (compared with the control, **P < 0.01, *P < 0.05) (scale bar: 50μM). F) Control siRNA- or P2Y2R siRNA-transfected THP-1 cells were treated with ATP (10 μM) or UTP (10 μM) for 24 h, and the activity of MMP-2 and MMP-9 in the CM was evaluated by gelatin zymography as described in the Methods.
Fig.5
Fig.5. Inhibition of P2Y2R reduced collagen crosslinking and LOX secretion in an in vivo mouse model
A) Athymic nude mice were divided into 2 groups and injected subcutaneously with MDA-MB-231-EV (n = 10) or MDA-MB-231-P2Y2R-shRNA (n = 10) (3 × 106 cells/100 μl of serum-free medium). MDA-MB-231-EV-injected or MDA-MB-231- P2Y2R-shRNA-injected animals were sacrificed at day 60, and blood was collected by heart puncture. Body weights and tumor volumes were measured every 3 days during tumor development. Body weight (B) and tumor volume (C) at the end of 60 days are presented (compared with the MDA-MB-231-EV-injected mice, *P < 0.05). D) LOX levels in blood serum were analyzed by Western blot analysis (compared with the MDA-MB-231-EV-injected mice, *P < 0.05). E) Lung tissue sections from MDA-MB-231-EV- and MDA-MB-231-P2Y2R-shRNA-injected mice were stained with Picrosirius Red to analyze crosslinked collagen. Recruited CD11b+-immunoreactive BMDCs near the sites of collagen cross-linkage were counterstained with anti-CD11b antibody. White arrows indicate representative crosslinked collagen fibers. Black arrows indicate recruited CD11b+ BMDCs (scale bar: 50 μm).
Fig.6
Fig.6. The proposed model for the role of P2Y2R in pre-metastatic niche formation and tumor metastasis
Tumor microenvironments such as hypoxia induce ATP release from cancer cells and P2Y2R expression in monocytes. ATP released from cancer cells can activate P2Y2R both in monocytes and cancer cells. In monocytes, P2Y2R activation by ATP induces monocyte recruitment toward the tumor and MMPs secretion, resulting in TAMs formation and enhanced tumor growth. In addition, P2Y2R activation by ATP induces HIF-1α, resulting in LOX release in cancer cells. Released LOX induces collagen crosslinking, leading to the recruitment of BMDCs and pre-metastatic niche formation.
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