Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells

Highlights

• Extracellular ATP induced large intracellular ATP increase in cancer cell lines.

• Extracellular ATP promoted cell survival and drug resistance in cancer cells.

• Fluorescent ATP was shown to be internalized by human lung cancer A549 cells.

• The ATP internalization in A549 cells is partially mediated by macropinocytosis.

• Extracellular ATP plays important roles in the Warburg effect/cancer metabolism.

Abstract

ATP plays central roles in cancer metabolism and the Warburg effect. Intratumoral ATP concentrations are up to 10⁴ times higher than those of interstitial ATP in normal tissues. However, extracellular ATP is not known to enter cancer cells. Here we report that human A549 lung cancer cells internalized extracellular ATP by macropinocytosis as demonstrated by colocalization of a nonhydrolyzable fluorescent ATP and a macropinocytosis tracer high-molecular-weight dextran, as well as by a macropinocytosis inhibitor study. Extracellular ATP also induced increase of intracellular ATP levels, without involving transcription and translation at significant levels, and cancer cells’ resistance to ATP-competitor anticancer drugs, likely through the mechanism of ATP internalization. These findings, described for the first time, have profound implications in ATP-sharing among cancer cells in tumors and highlight a novel anticancer target.

Introduction

Metabolic reprogramming, or deregulation of cellular energetics, is now recognized as a hallmark of cancer [1]. ATP production by highly upregulated glycolysis in cancer cells even when oxygen is abundant, a phenomenon known as the Warburg effect [2], [3], is an area of intensive investigation with improved but still incomplete understanding. Interpretations of the functional reasons for upregulated glycolysis in the presence of continued mitochondrial oxidative phosphorylation (OXPHOS) and the relationship between glycolytic synthesis of ATP and other metabolic intermediates in cancer cells are controversial and evolving [3], [4], [5], [6]. Being metabolically heterogeneous, some cancer cells in tumors make significantly less ATP than other cancer cells or even normoxic normal cells [3]. However, cancer cells appear to be able to obtain all the ATP they need regardless of oxygen status. This is puzzling unless previously unrecognized mechanisms for securing ATP exist.

One potential source of ATP for cancer cells is the extracellular ATP pool [7]. Interstitial ATP concentrations in normal tissues are in the range of 1–1000 nM [8], [9]. In contrast, intratumoral ATP levels, i.e., extracellular ATP levels inside tumors, have recently been measured in the range of several hundred μM or higher [10], [11], [12], [13], or 10³–10⁴ times of those in normal tissues. However, neither the source of the ATP nor the destination of the molecule is known. Earlier experimental evidence suggests uptake [14], [15], [16], [17], [18] and release of ATP in normal animal cells [19], [20], providing conceptual and biological basis for potential ATP uptake by cancer cells. However, the uptake of extracellular ATP by cancer cells has never been demonstrated except by artificial means [21].

In our recent anticancer therapeutic studies, we observed that extracellular ATP significantly increased intracellular ATP levels in A549 human lung cancer cells and rescued glucose-deprived cancer cells treated with glucose transporter 1 (GLUT1) inhibitor WZB117 [22]. However, extracellular ATP did not rescue A549 cells treated with paclitaxel, a drug with an ATP-independent anticancer mechanism [22]. One of the possible explanations for these results is that extracellular ATP is directly taken up by A549 cells and contributes to intracellular ATP concentration increase, playing a significant role in cancer cell growth and survival. Because ATP is charged and therefore hydrophilic, it cannot cross the plasma membrane by itself. However, no plasma membrane-associated ATP transporter has ever been found. Thus, we further speculated that the ATP uptake might be mediated by some types of endocytic processes, bypassing the problem. In the present study, we tested this hypothesis by studying ATP transport mechanisms with a nonhydrolyzable fluorescent ATP. Extracellular ATP concentrations in the reported intratumoral ATP range were used to mimic in vivo conditions. To identify the functional significance of the extracellular ATP-induced intracellular ATP increase, extracellular ATP-induced drug resistance was also studied. The use of a nonhydrolyzable fluorescent ATP for demonstrating ATP internalization and extracellular ATP-mediated drug resistance in cancer cells have never been reported. The findings of this study may significantly impact our interpretation of the Warburg effect, expand our knowledge of ATP/energy sharing among cancer cells, and highlight a new target for cancer treatment.