Introduction
Cell fusion is a normal physiological event that plays a critical role in fertilization, placentation, myogenesis, osteogenesis, wound healing and tissue regeneration [
1‐
4]. In addition, it is strongly suggested to be a tumor inception contributor, progression and heterogeneity [
5]. Several studies have demonstrated the presence of hybrid cells in human cancers, in some cases comprising up to 40% of tumors [
6]. Recently,
Gast et al. have shown that hybrid cells could be found in both human pancreatic ductal adenocarcinoma cells and in the circulatory system, where they were associated with a poor prognosis [
7]. Moreover, our team recently reported that the fusion of pre-malignant and malignant mesenchymal cells triggers a genomic instability that resembles the instability found in human sarcomas [
8], and also showed that hybrids made from cancer mesenchymal cells with non-cancerous partners gain invasiveness, giving rise to highly metastatic tumors [
8,
9].
Hybrids resulting from homotypic or heterotypic cancer cells fusion are known to exhibit features such as high aggressiveness, drug resistance, metastatic capabilities, and to facilitate tumor proliferation when compared to non-hybrid parental cells [
6‐
9]. While hybrids can inherit genetic, and thus phenotypic, features from their parents, they also develop their own identity as a result of the intense remodeling of their genome, epigenome and transcriptome [
10,
11]. However, it is still unclear how these major cellular changes drive the gain-of-functions observed in hybrid cells and which molecular mechanisms are specifically activated upon fusion to allow greater dissemination and/or growth abilities.
Like polyploid cells, hybrids host a higher DNA content and are larger in size than their euploid counterparts [
12]. This greater cell volume and chromosome number presumes higher metabolic needs to ensure the continuity of cell division, growth and all basic cellular functions [
13]. For example, hyperploid glioblastoma cells were shown to be more metabolically active than their euploid peers [
13]. Along with immune evasion, metabolic reprograming is now one of the main hallmarks of cancer cells, and cellular energetics are considered as core traits of tumors, playing a major role in cancer cell proliferation and metastatic spreading [
14,
15].
Cancer cells generally use glycolysis instead of oxidative phosphorylation (OXPHOS) despite having enough oxygen levels. This phenomenon, known as the “Warburg Effect” [
16], confers an advantage by increasing the level of non-oxidative ATP and generating intermediates that are important for cell growth and dissemination [
17,
18]. 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a master sensor of cellular energy and adaptor to metabolic stress in cancer cells [
19]. Previous studies have established that AMPK is related in a complex manner with other metabolic/energy pathways such as SIRT1, Akt, mTOR and PARPs [
19‐
21]. AMPK is generally downregulated in cancer cells, thereby favoring ATP-consuming mechanisms including proliferation [
22]. The activation of AMPK has been shown to induce mitochondrial biogenesis triggering an anti-Warburg [
23] and anti-proliferative [
21] effect in several types of cancer [
24].
Modifying the intracellular energy metabolism is thus critical for the growth and dissemination of cancer cells. While many reports agree that hybrids display greater pro-oncogenic potential, little is known regarding their metabolic status or the metabolic consequences of cell fusion. It is highly possible that, upon merging, hybrids undergo extensive metabolic rewiring and must adapt to find the right equilibrium to primarily sustain their needs and pursue their development. Nonetheless, it is also possible that these metabolic changes trigger a higher metabolic stress, while supporting the acquisition of novel functions.
We recently showed that cell fusion promotes tumor progression (notably metastatic spread) by using hybrids made from the spontaneous cell fusion of IMR90 E6E7 RST (malignant) and IMR90 E6E7 (pre-malignant) cells [
9]. In the present study we used this well characterized model of mesenchymal tumor progression to investigate changes in energy metabolism operating in hybrid cells, following the hypothesis that their aggressiveness may result from an increased metabolic stress altering the AMPK pathway. To address these questions, we measured their mitochondrial respiration, glycolytic activity, and evaluated the effect of AMPK activation on their growth and motility.
Discussion
Cell fusion can lead to the production of highly malignant harmful cells at various steps of the oncogenic process [
27,
28], representing a potential target for therapeutic intervention. However, to reach that goal, it is essential to characterize the biological changes occurring in hybrid cells compared to their parents and to decipher the signaling pathways that are crucial for their survival and growth. Until now, hybrids have been shown to undergo a complex reorganization of their genomic, epigenomic and transcriptomic content [
29], but no link has been established between these events and their higher pro-oncogenic functions.
To better characterize hybrid cells and formulate new hypothesis regarding their increased malignity, we performed gene expression profiling experiments using four metastatic hybrid cell lines (H1-H4) and their non-metastatic parental cells (i.e. pre-malignant E6E7 and malignant RST) [
9]. Interestingly, GO analysis of the most upregulated genes in hybrids vs. parents evidenced an enrichment in the pathways related to metabolism, especially energy metabolism. We thus explored the idea that, concurrently to their nuclear reprogramming, hybrid cells might undergo a remodeling of their energy traits to secure their growth and sustain their higher invasiveness.
In agreement with a higher metabolic imbalance in hybrids, we found that AICAR, a pharmacological agent targeting AMPK which is a major energy sensor in the cell, specifically blocked the proliferation of hybrids while having no effect on RST and E6E7 parental cells. In addition, AICAR attenuated the invasive processes in hybrids with enhanced glycolysis (i.e. H1, H2, H3, H5 and H6). As described above, H4 did not show evidence of glycolysis hyper-activation compared to RST and, hence, it is possible that AICAR only precludes, (or is most effective on) the invasive processes driven by glycolysis.
Altogether, our results highlight the anti-proliferative effect of AICAR on hybrid cells using cell counting by flow cytometry. This assay selects the cells based on their physical properties (size, granularity), and presents the advantage to be simple and straightforward when manipulating poorly characterized or complex cells (hybrids vs. non-hybrids).
Surprisingly, AICAR enhanced the invasive capacities of parental RST cells, perhaps due to the higher expression level of AMPK in these cells compared to hybrid cells. Previous studies have shown that AMPK can function as a tumor promotor under certain circumstances, notably when
PRKAA1, coding the catalytic subunit-alpha-1 of AMPK, is highly expressed [
30]. Moreover, it is important to consider that RST cells generate UPS-like tumors in vivo that lack many features of the corresponding human malignant tumors (i.e. no genetic instability and no metastatic capacities), contrary to hybrids (i.e. highly aneuploid and metastatic) [
9]. In that sense, RST cells do not really mirror the cancer cells found in human UPS whereas hybrid cells do, therefore identifying drugs able to disrupt the propagation of hybrids was the ultimate goal. Altogether, these results emphasize the value of treating hybrid cells with AICAR as previously reported for other aneuploid cancer cells [
31].
Interestingly, transcriptome analysis of parental and hybrid cells further revealed a specific decrease in the expression of the catalytic subunit-alpha-1 of AMPK (PRKAA1) in hybrid cells and no modification of the regulatory subunit (PRKAB1). Western blot confirmed a tendency of a decrease of AMPK alpha in hybrids with the limitation that the antibody was directed against alpha1 and alpha2. isoforms, and not solely against alpha1. AMPK is a major mediator of cellular energy homeostasis and its loss might thus reflect a selection pressure occurring specifically in hybrids, which presumably require more energy to survive. Indeed, analysis of the energy metabolic profiles of hybrid and parental cells (respiration rates and glucose consumption) revealed hybrids to be inclined to higher metabolic activities. Not only did they rely on a ‘Warburg-like’ metabolism like RST malignant cells (i.e. lower respiratory rate and higher glucose consumption), but also on greater metabolic flux. Indeed, five hybrids out six (i.e. H1, H2, H3, H5 and H6) used more glucose (significantly for H2 and H3) than RST cells, underlining their greater glycolytic rates.
Such increases might be necessary to mobilize sufficient amounts of nutrients and energy for anabolic reactions and growth [
32]. In this study, only H4 did not show higher glycolytic requirements. It still remains possible that this particular hybrid relies on other critical metabolic pathways for growth, such as glutamine metabolism [
33], but this would need further investigation. Moreover, corroborating these results, we found that hybrids up-regulated several genes of the glycolytic pathway at the mRNA level. Remarkably, many genes of glycolysis have been previously implicated in cancer progression [
34]. To sum up, our results revealed that hybrids are more metabolically active than their RST euploid counterpart, relying essentially on higher glycolytic rates (but also possibly on other pathways) to survive and proliferate. Interestingly, overactivating glycolysis leads to the production of lactate, an oncometabolite well known for its role in immune evasion and metastatic development, a characteristic recurrently found in H1 to H6 hybrid cells [
9,
34‐
37,
38].
The activation of AMPK was previously shown to trigger mitochondrial biogenesis and OXPHOS, causing anti-Warburg [
23] and anti-proliferative [
21] effects in several types of cancer [
24], such as leukemia [
39], breast cancer [
40], pancreatic cancer [
41], hepatocellular carcinoma [
42], and prostate cancer [
43]. Here, we show that AICAR, a direct AMPK activator, is a potent agent against aneuploid hybrid cells functioning at high metabolic rates. Interfering with the metabolic status of hybrids (using AICAR here) seems to revoke both their growth and invasive capacity. Thus, it is likely that the over-activated metabolism of hybrids is necessary for their proliferation and invasion properties, creating an interesting therapeutic target. It is to note, that our first attempts to use metformin, another potential activator of AMPK did not show any selectivity against hybrids. However, AMPK is only an indirect target of metformin [
20], which can explain this result. As described above, we recently proved that cell fusion of E6E7 and RST cells creates hybrids able to generate UPS-like tumors with all the clinical traits of their human counterparts [
8,
9]. Suspecting that human UPS cells may be the result of cell fusion events, we then attempted to treat them with AICAR. Interestingly, IB105, IB106 and the fusion IB105/106 cells were all highly sensitive to AICAR treatment, exhibiting a drastic decrease in proliferation and migration (except for IB106 that barely migrates) in presence of the drug.
Altogether, our findings show that cell fusion generates hybrid cells with greater metabolic needs compared to their parents and that this specific feature may represent their Achilles heel, paving the way for new therapeutic approaches to treat human pleomorphic sarcoma and eradicate these aggressive cancer cells.
Materials and methods
Cell lines and reagents
IB105, IB106 and IB105/106 sarcoma cell lines, as well as all the IMR90 fibroblasts (1/IMR90 E6E7 CFP Blast
R (i.e. E6E7), and 2/IMR90 E6E7 HRAS
G12V SmallT Tert DsRed Puro
R (i.e. RST)) were cultured in RPMI-1640 (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified CO
2 incubator. Sarcoma cells were generated in the laboratory [
44], while IMR90 cell lines were kindly given by M. Teichmann [
45]. The latter were generated according to the cell transformation model described by Hahn et al. [
46,
47]. AICAR was purchased from Sigma Aldrich (Ref. A9978, St. Luis, MO).
Hybrid cells selection
As previously published in [
9], DsRed, GFP and CFP parental cell lines were generated by lentiviral infection using pSD136-puromycin-DsRed, pER69-puromycin-GFP, pER167-Blasticidin-CFP plasmids, respectively. 150,000 cells of each parental cell line were seeded together in 6-well plates. Spontaneous hybrid cells were selected after 72 h of contact by cell sorting (IB105/106) or double antibiotic addition (puromycin/blasticidin for IMR90 E6E7/E6E7 RST). Resistant and double fluorescent clones (i.e. CFP/DsRed for H1 to H6, and GFP/DsRed for IB105/106) were amplified (Supplementary Fig.
1, A and B) for further genetic and cellular analyses. All hybrids were shown to result from fusion events, and harbored a highly rearranged genome, together with new migration capacities and aggressiveness in vivo [
9].
Gene expression profiling
Gene expression analysis was carried out using Agilent Whole human 44 K Genome Oligo Array (Agilent Technologies) according to the manufacturer’s protocol. All microarrays were simultaneously normalized using the Quantile algorithm. T-tests were performed using Genespring (Agilent Technologies) and
P-values were adjusted using the Benjamini-Hochberg procedure. The
P-value and fold change cut-off for gene selection were 0.001 and 2, respectively. Gene ontology (GO) analysis was performed to establish statistical enrichment in GO terms using Genespring (Agilent Technologies). Heatmap and boxplot were performed using with R (v3.6.2). The datasets generated and analyzed are available at the GEO,
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171471.
Cell proliferation assay
Cells were seeded in 96-well plates at a concentration of 2000 cells/well (6 wells/per cell line) and treated with AICAR (700 μM) or vehicle (H
2O) 24 h later. RPMI galactose medium was prepared as indicated in [
27]. The number of cells was evaluated by flow cytometry (FACS Calibur, BD Biosciences) at day 6. Data were analyzed using FlowJo (Tree Star, Celeza GmbH) and GraphPad (La Jolla, CA) software programs. For each cell line, the average number of cells present in AICAR-treated wells at day 6 was calculated and expressed as a percentage of growth compared to the corresponding control wells (average number of cells present in the wells of the untreated control = 100% of growth).
Migration assay
Migration assays were performed using the IncucyteS3 Live-cell analysis system (Essen BioScience, Hertfordshire, UK). Briefly, cells were seeded in 96-well ImageLock plates (3 × 104 cells for IB105 DsRed, IB106 GFP and 2 × 104 for IB105/106 hybrids) and treated 24 h later with vehicle or AICAR (700 μM) for 24 h (12 wells/condition for each cell line). After removal of AICAR, a strip of cells was removed from each well using the WoundMaker device following the manufacturers’ instructions. The plate was then placed in the IncucyteS3 machine (37 °C and 5% CO2) for 24 h. Images were taken every 4 h for 24 h with a 10x objective. Cell migration analysis was done using the Incucyte ZOOM software (Essen BioScience, Hertfordshire, UK) and expressed as percentage relative wound density (RWD (%)), a metric taking into account the spatial cell density in the wound area relative to the spatial cell density outside of the wound area at every time point. RWD values are self-normalizing for changes in cell density which may occur outside the wound due to cell proliferation and/or pharmacological effects. Data plotting and statistical analysis was performed with GraphPad (La Jolla, CA) software.
Invasion assay
Invasion was monitored using Matrigel-coated cell culture inserts containing 8.0 μm pore transparent positron emission tomography membranes (Fisher Scientific). AICAR (700 μM) or vehicle pre-treatments were done into 6-well plates seeded with 105 of parental or hybrid cells. After 24 h of contact, control and AICAR-treated cells were collected and added to 24-well Boyden chamber plates (25,000 cells/well) in presence of RPMI-1640 (Gibco, Invitrogen) plus 5% FBS (upper chamber). The lower chamber was filled with RPMI-1640 plus 10% FBS to create a gradient supporting cell invasion. After 18 h of incubation, cells located at the top side of the membrane were removed using cotton-tipped swab, while invasive cells located at the other end were fixed with cold ethanol absolute for 15 min and stained with Hoechst 33,342 (1/5000) for 10 min at room temperature. For each insert the entire bottom membrane was acquired using an Axio Vert.A1(ZEISS) microscope. Quantification was done using the Image J cell counter plugin. Data were normalized according to the number of cells at time zero and 18 h evaluated in parallel (in mirror 24-well plates) by flow cytometry and plotted using GraphPad (La Jolla, CA) software.
Glucose quantification
5 × 10
3 cells of E6E7, RST and H1-H6 hybrids were seeded in 24-well plates. An aliquot of cell culture supernatant was removed every 24 h from day 0 to day 4 and kept for evaluation of glucose. The number of cells in the corresponding wells was determined each day by flow cytometry. Doubling times were then calculated using exponential curves in Excell software. Glucose was measured enzymatically in protein free extracts by spectrophotometric determination according to Bergmeyer [
48]. Data were plotted to generate glucose consumption curves. Linear equations were then defined for each cell line using Excell software and used to determine the quantity of glucose used produced by each line per doubling time.
Respiratory rate measurement
Oxygen consumption was measured at 37 °C using a Clark-type electrode (Oxygraph, Oroboros Instruments). The cells were pelleted at 1200 rpm for 5 min, resuspended in 200 μl of RPMI 1640 and added to the measurement chambers pre-filled with 2.1 ml of RPMI-1640/Hepes (10 mM) medium. Basal respiration was recorded, after stabilization of the electrode, in presence of 5 mM pyruvate for 10–15 min. Sequential additions of oligomycin (0,25 μg/ml) or dinitrophenol (DNP, 50-100 μM) were then added to monitor minimal (without oxidative phosphorylation) and maximal respiration rate, respectively. Citrate synthase activity was assayed according to the method described by Srere (Faloona and Srere, 1969) for normalization of the data. The reaction mixture contained 50 mM Tris buffer (pH 7.5), 0.1 mM EDTA, 0.1 mM acetyl-CoA, 0.2 mM 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 0.05% triton X-100, and the enzyme extract. The reaction was initiated with 0.5 mM oxaloacetate and monitored at 412 nm for 10–15 min at 25 °C. The enzyme activity was calculated using an extinction coefficient of 13,600 M− 1 cm− 1 at 412 nm.
Western blot
Protein extracts were prepared as described in [
49]. AMPKα protein expression level was evaluated using a primary antibody directed against AMPKα (Cell signaling technology #2532). This antibody recognizes alpha 1 and alpha 2 isoforms of the catalytic subunit, but not the regulatory beta subunit. 20
μ g of proteins were loaded on the gel and separated by SDS-page. After the transfer onto a PVDF membrane, the membrane was blocked in PBS-Tween 0.1% BSA 5% buffer and then incubated with the primary antibody (1:1000) overnight at 4 °C. After several washes, the membrane was then incubated with a horseradish-peroxidase-linked anti-rabbit antibody (Cell signaling technology #12–2018) for 1 h at room temperature. Signal was detected using PXi (Syngene, Cambridge, UK) after incubation with chemiluminescent substrate (ECL Immobilon Western, WBKLS0100, Merck, Darmstadt, Germany). β-actin (Sigma #A5316, 1:10000, 1 h at room temperature) with an anti-mouse secondary antibody (Cell signaling technology #08–2018; 1:10000, 1 h at room temperature) was used as a loading control for quantification.
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