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Extracellular Vesicles Derived from Metastatic Melanoma Cells Transfer a7-nAChR mRNA, Thus Increasing the Surface Expression of the Receptor and Stimulating the Growth of Normal Keratinocytes
M. L. Bychkov1, А. V. Kirichenko1,2, I. N. Mikhaylova3, A. S. Paramonov1, М. P. Kirpichnikov1,4, М. А. Shulepko1, E. N. Lyukmanova1,4*
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
2Moscow Institute of Physics and Technology, State University, Dolgoprudny, Moscow region, 141701 Russia
3Federal State Budgetary Institution named N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Healthcare of the Russian Federation, Russia, Moscow, 115548 Russia interdisciplinary Scientific and Educational School of Moscow University "Molecular Technologies of the Living Systems and Synthetic Biology", Faculty of Biology, Lomonosov Moscow State University, Moscow, 119234 Russia *E-mail: [email protected] Received April 29, 2022; in final form, July 27, 2022 DOI: 10.32607/actanaturae.11734
Copyright © 2022 National Research University Higher School of Economics. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT We have previously shown that extracellular vesicles secreted by metastatic melanoma cells stimulate the growth, migration, and sternness of normal keratinocytes. This study showed for the first time that extracellular vesicles secreted by the metastatic melanoma cell lines mel H, mel Kor, and mel P contain, both at the mRNA and protein levels, the a7-type nicotinic acetylcholine receptor (a7-nAChR), which is involved in the regulation of the oncogenic signaling pathways in epithelial cells. Incubation with the vesicles secreted by mel H cells and containing the highest amount of mRNA coding a7-nAChR increased the surface expression of a7-nAChR in normal Het-1A keratinocytes and stimulated their growth. Meanwhile, both of these effects disappeared in the presence of a-bungarotoxin, an a7-nAChR inhibitor. A bioinformatic analysis revealed a correlation between the increased expression of the CHRNA7 gene coding a7-nAChR in patients with metastatic melanoma and a poor survival prognosis. Therefore, extracellular vesicles derived from metastatic melanoma cells can transfer mRNA coding a7-nAChR, thus enhancing the surface expression of this receptor and stimulating the growth of normal keratinocytes. Targeting of a7-nAChR may become a new strategy for controlling the malignant transformation of keratinocytes. KEYWORDS a7-nAChR, vesicles, metastatic melanoma, keratinocytes, oncotherapy, cancer. ABBREVIATIONS a7-nAChR - a7 nicotinic acetylcholine receptor; BEBM - bronchial epithelial cell growth basal medium; a-Bgtx - a-bungarotoxin; HRP - horseradish peroxidase; WST-1 - water-soluble tetrazolium salt 1.
INTRODUCTION
Melanoma is an aggressive tumor that is formed by transformed melanocytes [1]. Melanoma progression is mediated by the secretion of extracellular vesicles (membrane-enveloped structures loaded with various proteins and nucleic acids) by tumors cells. Extracellular vesicles are involved in the transduc-
tion of oncogenic signals between tumor cells, as well as between the tumor and the surrounding tissues [2, 3]. Fibroblasts, immune cells, and keratinocytes regulate melanocyte physiology and control melanoma proliferation, invasion, and angiogenesis by the secretion of paracrine growth factors and intercellular communication [4, 5]. However, keratinocytes
can secrete mitogenic and pro-inflammatory factors under stress conditions (e.g., under photo-induced damage) [6].
We have shown previously that extracellular vesicles secreted by metastatic melanoma cells stimulate the growth, migration, and stemness of normal ke-ratinocytes [7]. The a7 nicotinic acetylcholine receptor (a7-nAChR) is involved in the regulation of the differentiation and growth of normal keratinocytes [8]. Its activation by nicotine or nicotine derivatives contained in tobacco (nitrosamines) promotes malignant transformation of keratinocytes [9]. However, the potential involvement of a7-nAChR in the stimulation of the keratinocyte growth induced by extracellular vesicles derived from melanoma cells has not been studied yet.
Here, we demonstrated for the first time that extracellular vesicles secreted by metastatic melanoma cells contain a7-nAChR at the mRNA and protein levels. Incubation in the presence of vesicles derived from the mel H cells increased the surface expression of a7-nAChR in normal keratinocytes and stimulated their growth; these effects were not observed in the presence of a-bungarotoxin (a-Bgtx), an a7-nAChR inhibitor. These findings provide a new insight into the role of extracellular vesicles secreted by metastat-ic melanoma and a7-nAChR in the malignant transformation of keratinocytes.
EXPERIMENTAL
The metastatic melanoma cell lines mel H, mel Kor, and mel P were collected from patients at the N.N. Blokhin National Medical Research Center of Oncology, Ministry of Healthcare of the Russian Federation (Moscow, Russia), and characterized earlier [10]. The cells were grown in the RPMI-1640 medium (PanEco, Russia) supplemented with 10% fetal bovine serum (Cytiva, UK) and 1% penicillin/streptomycin (PanEco). To remove endogenous exosomes, fetal bovine serum was centrifuged (70 min, 120,000 g),
filtered, and mixed with cell media. Human keratinocytes Het-1A (ATCC, USA) were cultured in the BEB medium (Lonza, Switzerland) according to the procedure described earlier [7]. Extracellular vesicles were isolated from metastatic melanoma cells using the procedure described in [7]: the cells were cultured in an exosome-depleted medium; the growth medium was centrifuged sequentially at 10,000 g (15 min, 4°C) and 120,000 g (70 min, 4°C). Protein complexes were removed by gel filtration using the Superdex G-250 resin (GE Healthcare, USA). Vesicle size was estimated by the dynamic light scattering (DLS) method using the DynaPro Titan instrument (Wyatt Technology, USA). Expression of the exosomal marker TSG101 in the vesicles was confirmed by Western blotting.
The nAChR subunit mRNA expression was analyzed by real-time PCR according to the procedure described earlier in [7]. Expression of the CHRNA3, CHRNA4, CHRNA5, CHRNA7, CHRNA9, CHRNB2, and CHRNB4 genes (primers are listed in Table 1) was analyzed using a Roche LightCycler 96 amplifi-cator (Roche, Switzerland). The mRNA level was normalized to the expression of S18 ribosomal RNA.
The presence of a7-nAChR in the extracellular vesicles at the protein level was analyzed by Western blotting [7]. After the gel electrophoresis and transfer of vesicle lysates, nitrocellulose membranes were blocked with 5% milk and incubated with primary anti-TSG101 (1 : 1000, ABIN2780037, Antibodies-Online, Germany) or anti-a7-nAChR rabbit antibodies (1 : 1000, ABIN5611363, Antibodies-Online) at 4°C overnight, washed, and incubated with HRP-conjugated anti-rabbit antibodies (1 : 5000, 111-035-003, Jackson Immunoresearch, USA) for 1 h at 20°C. The membranes were then washed, and the HRP signal was registered using the ECL substrate (Bio-Rad, USA) and an ImageQuant LAS 500 camera system (GE Healthcare, USA).
To study the effect of extracellular vesicles on keratinocyte proliferation, the cells were seeded in
Table 1. The primers used in this study
Gene Primer Amplicon size, bp
Forward Reverse
S18 SSU RNA CTC AAC ACG GGA AAC CTC AC CGC TCC ACC AAC TAA GAA CG 110
CHRNA3 TGT CCC TCT CTG CTT TGT CAC CCC AGG TTC TTG ATC GGA TGT T 169
CHRNA4 TCG TCC TCT ACA ACA AGT GAG GGT CCA GGA GCC GAA TTT CA 199
CHRNA5 CGT CTG GTT GAA ACA GGA ATG G ACA GTG CCA TTG TAC CTG ATG A 185
CHRNA7 TTT ACA GTG GAA TGT GTC AGA TGT GGA ATG TGG CGT CAA G 88
CHRNA9 GGA GGC CAG ACA TCG TCT TA CAC TGC TGG TTG TCA AAA GGG 168
CHRNB2 ATC TCC TGG ATC CTT CCC GC AGA AGG ACA CCT CGT ACA TGC C 290
CHRNB4 CGC CTT CCC TGG TCC TTT TC TGT TCA CAC CCT CGT AGC GG 381
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Fig. 1. Analysis of the a7-nAChR expression in extracellular vesicles derived from metastatic melanoma cells. (A) - Analysis of the CHRNA7 expression in extracellular vesicles derived from mel H, mel Kor, and mel P cells. Expression of mRNA was assayed by real-time PCR and normalized to the S18 ribosomal RNA. The data are presented as the mean mRNA level ± SEM (n = 4). ** (p < 0.01) indicates a significant difference between the data groups according to one-way ANOVA, followed by the Tukey's post hoc test. (6) - Analysis of a7-nAChR protein expression in extracellular vesicles derived from mel H, mel Kor, and mel P cells by Western blotting. TSG101 was used as an exosomal marker
B
96-well plates (5 x 103 cells/well); after 24 h, they were supplemented with vesicles (total protein concentration 50 ^g/ml) and/or 10 ^M a-bungarotoxin (a-Bgtx, an a7-nAChR inhibitor, Tocris, UK) and additionally incubated for 72 h without media replacement. The concentration of the total vesicular protein corresponded to that in the plasma of the cancer patients (20-100 ^g/mL) [7]. Cell viability was analyzed using a WST-1 colorimetric assay (Santa Cruz, USA) [11]. The data were normalized to averaged data in the control wells containing untreated cells.
The effect of the vesicles and a-Bgtx on a7-nAChR expression in keratinocytes was studied after staining the cells with TRITC-labeled a-Bgtx (Sigma-Aldrich, USA) using an Attune NxT flow cytometer (Life Technologies, USA) and the procedure described earlier [11]. The median fluorescence was normalized to the autofluorescence of unstained cells.
The correlation between the CHRNA7 expression level in patients with metastatic melanoma from the TCGA database (the SKCM study) and the prognosis of their survival was analyzed using the Xena software [12].
RESULTS AND DISCUSSION
Extracellular vesicles secreted by melanoma cells contain microRNA, mRNA, and proteins that stimulate the proliferation, migration, and stemness of normal keratinocytes [7]. However, the recruitment of nAChRs, which regulate many oncogenic processes in epithelial cells, into these effects of extracellular vesicles has not been studied previously.
We have demonstrated by real-time PCR for the first time that extracellular vesicles secreted by patient-derived metastatic melanoma cells mel H, mel Kor, and mel P contain CHRNA7 mRNA encoding the a7-nAChR subunit of the homopentameric receptor (Fig. 1A). The highest CHRNA7 expression level was observed for vesicles secreted by mel H cells. Meanwhile, no mRNA encoding the a3, a4, a5, a9, |32, and |4 subunits of nAChR were detected. Western blotting proved that the vesicles derived from all the studied melanoma cell lines contained the a7-nAChR protein (Fig. 1B). Interestingly, the previous analysis of the protein composition of extracellular vesicles secreted by primary melanomas had detected no a7-nAChR [3]. Expression of this receptor can possibly be a specific feature of extracellular vesicles derived from metastatic melanoma.
We have shown earlier that extracellular vesicles secreted by metastatic melanoma mel P contain mRNA encoding the epidermal growth factor receptor (EGFR), and that incubation of normal keratinocytes in the presence of these vesicles leads to upregulat-ed EGFR expression on the keratinocyte surface and stimulates their proliferation [7]. Here, we studied the effect of extracellular vesicles derived from metastat-ic melanoma mel H, mel Kor, and mel P cells on the a7-nAChR expression in normal keratinocytes. Flow cytometry revealed that only incubation in the presence of extracellular vesicles derived from mel H cells causes a statistically significant upregulation of the a7-nAChR expression on the surface of normal ke-ratinocytes. Treatment of keratinocytes with vesicles
Fig. 2. Analysis of the effects of extracellular vesicles secreted by metastatic melanoma cells and a-Bgtx on the a7-nAChR expression and keratinocyte proliferation. A—C - Expression of a7-nA-ChR on the surface of normal keratinocytes incubated in the presence of extracellular vesicles derived from mel H (A), mel Kor (B), and mel P (C) cells and/or a-Bgtx. The data are presented as normalized median fluorescence (MFI) ± SEM (n = 3). * (p < 0.05) and ** (p < 0.01) indicate a significant difference between the data groups according to one-way ANOVA, followed by the Tukey's post hoc test. D—F - The effects of extracellular vesicles derived from mel H (D), mel Kor (E), and mel P (F) cells and/or a-Bgtx on the proliferation of normal keratinocytes. The data are % of untreated cells ± SEM (n = 4). # (p < 0.05) indicates a significant difference from the untreated cells according to the one-sample t-test. * (p < 0.05) indicates a significant difference between the data groups according to one-way ANOVA, followed by the Tukey post hoc test
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derived from mel Kor and mel P had no effect on the expression level of the receptor (Fig. 2A—C). The results are consistent with the PCR data showing that the highest CHRNA7 expression level is actually observed in vesicles derived from mel H cells (Fig. 1A). It is plausible that vesicles derived from metastat-ic melanoma cells mel H transfer mRNA encoding a7-nAChR to keratinocytes, thus increasing the expression of this receptor in normal cells. Interestingly, incubation in the presence of a-Bgtx, an inhibitor of a7-nAChR, reduced the expression of this receptor on the keratinocyte surface (Fig. 2A—C) both in the
presence and absence of vesicles derived from mel H cells, pointing to some positive feedback between the receptor activity and its expression.
In all the cases, incubation with vesicles derived from the mel H, mel Kor, and mel P cells significantly increased the number of viable keratinocytes (Fig. 2D-F). However, a-Bgtx cancelled the mitogen-ic effect induced only by mel H-derived vesicles and this correlates with the fact that incubation of kerati-nocytes with vesicles from mel Kor and mel P caused no changes in the a7-nAChR expression in keratinocytes (Fig. 2B,C). It is noteworthy that incubation
CHRNA7
— below median (n = 176)
— above median (n = 176)
: 0.003
2500
5000 7500 Days
10000
Fig. 3. Bioinformatic analysis of the correlation between the survival of patients with metastatic melanoma and the CHRNA7 expression level. Patients were divided into two groups with the gene expression levels above and below the median value. Statistical analysis of patient survival was performed by the Kaplan-Meier method using the log-rank test
with a-Bgtx in the absence of vesicles did not significantly reduce the number of viable keratinocytes (Fig. 2D-F), although the toxin significantly reduced the expression of the receptor (Fig. 2A—C). This indicates that keratinocyte growth is independent of the a7-nAChR regulation under normal conditions, but transfer of the CHRNA7 gene by the vesicles from mel H cells significantly increases the receptor expression in keratinocytes, thus additionally stimulating their proliferation. It seems that, although expression of the a7 receptor is comparable in all the analyzed types of vesicles (Fig. 1B), CHRNA7 mRNA is the principal transferred component that stimulates keratinocyte growth in the presence of vesicles. Other factors unrelated to a7-nAChR (e.g., EGFR mRNA)
are probably responsible for the increased keratino-cyte proliferation observed upon incubation with vesicles from the mel Kor and mel P cells [7].
In order to understand how the CHRNA7 expression level can affect the development of oncogenic processes and, particularly, correlate with cell malignancy, we performed a bioinformatic analysis of the expression of this receptor in biopsy specimens taken from patients with metastatic melanoma. A KaplanMeier analysis showed that the upregulated CHRNA7 expression icorrelates with an unfavorable survival prognosis in patients with metastatic melanoma (Fig. 3). Our findings indicate that a7-nAChR is potentially involved in the pathogenesis of metastatic melanoma, and that transfer of mRNA encoding this receptor within extracellular components can be a mechanism responsible for the stimulation of tumor progression.
CONCLUSIONS
Expression of a7-nAChR, both at the mRNA and protein levels, was detected for the first time in extracellular vesicles secreted by different lines of meta-static melanoma cells. Extracellular vesicles derived from the mel H cells demonstrating the highest CHRNA7 expression were shown to transfer receptor mRNA to normal keratinocytes, thus increasing the a7-nAChR expression on their surface and stimulating their growth. Since no such effect of vesicles derived from the mel H cells was observed in the presence of a-Bgtx, it is a promising strategy to target a7-nAChR to control the malignant transformation of normal keratinocytes.
This work was supported by the Russian Science Foundation (project No. 17-74-20161).
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