Expression Pattern of Purinergic Signaling Components in Colorectal Cancer Cells and Differential Cellular Outcomes Induced by Extracellular ATP and Adenosine.

Annette Larsen,Alexandre Escargueil,Clémentine Dillard,Elodie Pramil,Michèle Sabbah,Laurent Jourdren,Ammara Mohammad,Virginie Puchois,Vincent Maréchal,Chloé Borde

doi:10.3390/ijms222111472

Abstract

The purine nucleotide adenosine triphosphate (ATP) is known for its fundamental role in cellular bioenergetics. However, in the last decades, different works have described emerging functions for ATP, such as that of a danger signaling molecule acting in the extracellular space on both tumor and stromal compartments. Beside its role in immune cell signaling, several studies have shown that high concentrations of extracellular ATP can directly or indirectly act on cancer cells. Accordingly, it has been reported that purinergic receptors are widely expressed in tumor cells. However, their expression pattern is often associated with contradictory cellular outcomes. In this work, we first investigated gene expression profiles through “RNA-Sequencing” (RNA Seq) technology in four colorectal cancer (CRC) cell lines (HT29, LS513, LS174T, HCT116). Our results demonstrate that CRC cells mostly express the A2B, P2X4, P2Y1, P2Y2 and P2Y11 purinergic receptors. Among these, the P2Y1 and P2Y2 coding genes are markedly overexpressed in all CRC cells compared to the HCEC-1CT normal-like colonic cells. We then explored the cellular outcomes induced by extracellular ATP and adenosine. Our results show that in terms of cell death induction extracellular ATP is consistently more active than adenosine against CRC, while neither compound affected normal-like colonic cell survival. Intriguingly, while for the P2Y2 receptor pharmacological inhibition completely abolished the rise in cytoplasmic Ca2+ observed after ATP exposure in all CRC cell lines, Ca2+ mobilization only impacted the cellular outcome for HT29. In contrast, non-selective phosphodiesterase inhibition completely abolished the effects of extracellular ATP on CRC cells, suggesting that cAMP and/or cGMP levels might determine cellular outcome. Altogether, our study provides novel insights into the characterization of purinergic signaling in CRC.

Highlights

In addition to its fundamental role in cellular bioenergetics, the purine nucleotide adenosine triphosphate (ATP) plays a crucial role in the extracellular space as a signaling molecule [1,2]
Because 2D cell cultures have limitations that might impact the response to tested compounds, we evaluated the capability of both extracellular ATP and adenosine to inhibit the growth of 3D tumor cell spheroids, which are believed to more closely mimic in vivo conditions (Figure 3B)
While pharmacological inhibition of the P2Y2 receptor completely abolished the rise of cytoplasmic Ca2+ induced by ATP for our four colorectal cancer (CRC) cell lines (Figure 6A), the co-incubation of AR-C 118925XX with extracellular ATP only impacted the cellular outcome of HT29 cells (Figures 6B and S4). These results suggest that, except for HT29 cells, neither P2RY2 nor rapid Ca2+ mobilization is likely to play a major role in extracellular ATP signaling leading to cell death induction in CRC cells

Summary

Introduction

In addition to its fundamental role in cellular bioenergetics, the purine nucleotide adenosine triphosphate (ATP) plays a crucial role in the extracellular space as a signaling molecule [1,2]. ATP is actively released in the pericellular environment in response to several stimuli, including (1) inflammation-related biological processes, (2) cellular stress and tissue damage during tumorigenesis, (3) cells undergoing apoptosis, and (4) exosomes secreted by cancer cells [3,4,5]. Extracellular ATP can be secreted during the process of immunogenic cell death induced by chemotherapeutics or released during necrosis [6,7]. This accumulation of extracellular ATP facilitates the recruitment of macrophages and dendritic cells (DCs) by acting as a potent chemotactic stimulus [7,8]. The membrane-bound CD73 (or ecto-50 nucleotidase) is considered as the rate limiting enzyme in the production of extracellular adenosine [13]

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Results

Conclusion