Exosome-mediated transfer of miR-222 is sufficient to increase tumor malignancy in melanoma
- Federica Felicetti†1,
- Alessandra De Feo†1,
- Carolina Coscia1,
- Rossella Puglisi1,
- Francesca Pedini1,
- Luca Pasquini1,
- Maria Bellenghi1,
- Maria Cristina Errico1,
- Elena Pagani2 and
- Alessandra Carè1Email authorView ORCID ID profile
© Felicetti et al. 2016
Received: 9 July 2015
Accepted: 9 February 2016
Published: 24 February 2016
Growing evidence is showing that metastatic cell populations are able to transfer their characteristics to less malignant cells. Exosomes (EXOs) are membrane vesicles of endocytic origin able to convey their cargo of mRNAs, microRNAs (miRs), proteins and lipids from donors to proximal as well as distant acceptor cells. Our previous results indicated that miR-221&222 are key factors for melanoma development and dissemination. The aim of this study was to verify whether the tumorigenic properties associated with miR-222 overexpression can be also propagated by miR-222-containing EXOs.
EXOs were isolated by UltraCentrifugation or Exoquick-TC® methods. Preparations of melanoma-derived vesicles were characterized by using the Nanosight™ technology and the expression of exosome markers analyzed by western blot. The expression levels of endogenous and exosomal miRNAs were examined by real time PCR. Confocal microscopy was used to evaluate transfer and uptake of microvesicles from donor to recipient cells. The functional significance of exosomal miR-222 was estimated by analyzing the vessel-like process formation, as well as cell cycle rates, invasive and chemotactic capabilities.
Besides microvesicle marker characterization, we evidenced that miR-222 exosomal expression mostly reflected its abundance in the cells of origin, correctly paralleled by repression of its target genes, such as p27Kip1, and induction of the PI3K/AKT pathway, thus confirming its functional implication in cancer. The possible differential significance of PI3K/AKT blockade was assessed by using the BKM120 inhibitor in miR-222-transduced cell lines. In addition, in vitro cultures showed that vesicles released by miR-222-overexpressing cells were able to transfer miR-222-dependent malignancy when taken-up by recipient primary melanomas. Results were confirmed by antagomiR-221&222 treatments and by functional observations after internalization of EXOs devoid of these miRs.
All together these data, besides generally confirming the role of miR-222 in melanoma tumorigenesis, supported its responsibility in the exosome-associated melanoma properties, thus further indicating this miR as potential diagnostic and prognostic biomarker and its abrogation as a future therapeutic option.
Keywords(da 3 a 10): Melanoma MicroRNA Exosomes miR-222 PI3K/AKT
Malignant melanoma is the most aggressive form of skin cancer whose incidence is doubling almost every 10 years. Although surgical resection is mostly a definitive treatment at the primary stages of the disease, the survival of patients is strongly reduced after metastatic dissemination, mostly due to resistance to conventional therapies [1, 2]. Therefore it is important to use new molecular approaches in order to better understand the mechanisms underlying melanoma progression.
MicroRNAs (miRs) are non-coding RNAs that regulate gene expression mostly at post-transcriptional level playing important roles in nearly all the biological functions, including tumor development and dissemination . These small non coding RNAs have revealed a great potential as early diagnosis markers being highly stable and able to discriminate different subtypes of cancer . Moreover the profile of circulating miRs was shown to reflect the expression pattern of tumor tissues .
Our previous results showed that miR-221&222 are key factors for melanoma development and dissemination, as they control the progression of the neoplasia through the down-modulation of several direct targets, as p27Kip1/CDKN1B, c-KIT receptor and c-FOS, all playing antineoplastic functions. Accordingly the miR-221&222-dependent repression leads to enhanced proliferation as well as differentiation and apoptosis blockade in melanoma cells [6, 7].
Growing evidence is showing that miRs are not strictly cellular, but are secreted in extracellular compartments, as body fluids as well as cell culture media, where they might be transported by vesicle and non vesicle carriers, including “exosomes” (EXO) . EXOs are nanovesicles of diameter ranging between 50 to 140 nm, distinguished from other cell-derived vesicles by their origin, size, morphology and composition . In fact, due to their endosomal origin, EXOs are enriched of certain proteins, including members of the tetraspanin family (CD9, CD81, CD82, CD63), heat shock proteins (Hsp60, Hsp70, Hsp90) and proteins of the multivesicular bodies (annexins, Rab GTPases and endosomal sorting complexes required for transport (ESCRT) proteins) . EXOs secreted by tumor cells were shown to transfer oncogenic properties via horizontal propagation of mRNAs, miRs and proteins. More important, upon their release in the extracellular environment, EXOs are utilized by tumors for both local and distant cellular communications, as these nanoparticles are able to transfer their cargo into the acceptor cells in autocrine and paracrine fashions .
In this study, we evaluated whether the presence of increased levels of miR-222 into melanoma-released EXOs were able to transfer the aggressive behavior of the donors into the acceptor cells. We also evaluated the possible functional role of some miR-222-related molecules carried by purified EXOs in supporting melanoma malignancy.
Human melanoma cell lines were stabilized from surgical specimens obtained from primary or metastatic tumors at Istituto Nazionale Tumori (Milan, Italy). Cell lines were characterized for growth in soft agar and, whenever possible, their metastatic potential was evaluated into athymic nude mice. Early passages cells were obtained from bioptic specimens at Istituto Dermopatico dell’Immacolata (Rome, Italy). All biological materials were obtained with the informed consent of patients and the study was conducted according to the Declaration of Helsinki Principles. An institutional approval for the performed experiments was not required. The cell lines were authenticated according to standard short tandem repeat (STR)-based genotyping. Melanoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO by Life Technologies, Paisley, UK) supplemented with 10 % FBS (GIBCO). Cells were incubated at 37 °C and supplemented with 5 % CO2 in humidified chamber. When indicated, melanoma cell lines were lentivirally transduced with miR-222 as previously reported [6, 7, 12]. Treatments with NVP-BKM120 (Selleckchem, Houston, TX, USA), a PI3K specific pan inhibitor, were performed at doses ranging between 1 and 5 µM in synchronized or not synchronized melanoma cells, in presence of 5 or 10 % FBS previously deprived of endogenous microvesicles by ultracentrifugation.
Exosome isolation and tracking analysis
EXOs were isolated from 24 h cell culture media by ultracentrifugation (UC) or Exoquick-TC (EQ) (System Biosciences, Mountain View, CA, USA) methods according to standards procedures or manufacturer’s instruction, with minor modifications (Fig. 1a). For exosome purification, serum was depleted of bovine EXOs by ultracentrifugation at 100,000×g for 6 h, followed by passing it through 0.2 µm filter prior to use. The protein concentration of EXOs was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA) and in some cases the number and size of EXOs were directly tracked using the Nanosight NS300 system (Nanosight™ technology, Malvern, UK), configured with a 488 nm laser and a high-sensitivity sCMOS camera. Videos were collected and analyzed using the NTA software (version 3.0). For each sample, multiple videos of 60 s duration were recorded generating replicate histograms that were averaged.
Exosome labeling and internalization
Cells were labeled by including into the culture medium (DMEM supplemented with 0.3 % FBS-UC) the fatty acid molecule BODIPY® FL C16. After 5 h of incubation, the dye in excess was washed out and cell culture media containing EXOs (with fluorescent phospholipids incorporated into membranes) were recovered. Thirty micrograms of EXOs (purified with EQ methods) were co-cultured with 2 × 104 recipient cells grown in eight-well chamber slides (IBIDI, Martinried, Germany). After 2–3 h of incubation, cells were fixed in 4 % w/v paraformaldehyde (Sigma–Aldrich) for 10 min. Next, cells were stained by Alexa Fluor 647 conjugate phalloidin (Immunological Sciences, Rome, Italy) and nuclei by Hoechst 333258 dye (Sigma–Aldrich). Exosomal and cellular staining were analyzed by Olympus FV-1000 laser-scanning confocal microscopy.
In vitro experimental model of fusion and functional assays
The same amounts of EXO/Tween or EXO/miR-222 purified EXOs, recovered from conditioned media of melanoma cells transduced either with Tween empty vector or miR-222, were incubated with recipient cells for 30 min at 37 °C before performing expression studies and functional assays. Vesicle preparations were used immediately after isolation. Invasion and chemotaxis studies were performed according to standard procedures . For tube formation assays, melanoma cells after being fused with EXOs were seeded into culture slide wells coated with 100 mg/cm2 of Matrigel growth factor reduced (Becton–Dickinson, Bedford, MA, USA). Tube-like formations defined as ≥2 cells forming elongated structures were counted after 24–48 h of incubation by microscope (JULI microscopy, Twin Helix, MI, Italy) at 10 × magnification from four different fields for each condition. Tube formation was analyzed manually  and by the Image J software. Experiments were conducted at least three times.
MiR-221 and miR-222 silencing by antagomir treatment
Chemically modified antisense oligonucleotides (antagomir or αmiR) were used to inhibit miR expression . The sequences of αmiR-221 and αmiR-222 used are: 5′P-GAAACCCAGCAGACAAUGUAGCU-3′-Chl and 5′P-GAGACCCAGUAGCCAGAUGUAGCU-3′-Chl, respectively; all the bases were 2′OMe modified. Antagomir oligonucleotides, were transfected at 200 nmol/L by using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s procedures. As a control, an unrelated antagomir, specifically the antagomir targeting miR-133a that is not expressed in melanomas (αmiR-133) was transfected as well. EXOs were purified from conditioned media 24 h after transfections.
Cell cycle analysis
Cell cycle analysis was performed in synchronized or not synchronized melanoma cells. In the first case cells were synchronized by the addition of Hydroxyurea (HU), final concentration 2 mM, per 16 h. Cultures were then washed and medium replaced. From this point, considered as t = 0, cells were monitored while they proceed along the cell cycle after specific treatments (i.e., EXO internalization or BKM120 supplementation). In not synchronized experiments, cells were seeded at roughly 60–70 % confluence and treated as indicated in DMEM supplemented with 5 % FBS in triplicate. Cells were collected, washed in PBS, and suspended in propidium iodide (PI) staining buffer (PBS containing 1 % Triton X-100, 50 mg/ml PI and 50 mg/ml RNase). Cells were then incubated for 30 min (37 °C) and DNA content measured by flow cytometry using a BD FACS Canto cytometer (BD Biosciences, CA, USA).
Western blot analysis was performed according to standard procedures. Exosome samples were lysed in buffer (0.5 % Triton; 300 mM NaCl; 50 mM TrisNaCl) supplemented with protease inhibitor cocktail. Antibodies listed below were used in accordance to the manufacturer’s instructions: CD63 (SBI System Biosciences, Mountain View, CA, USA), RAB5B, TSG101, HSP90 and CycD1 (Santa Cruz Biotechnology Dallas, TX; USA), LAMP2 and CAV-1 (BD Biosciences, CA, USA), RAB27A (Abnova, Taipei City, Taiwan), p85β (Abcam, Cambridge, MA, USA), AKT, ph-AKTSer473 and p27Kip1 (Cell Signaling, Beverly, MA, USA). β-ACTIN (Oncogene Research, La Jolla, CA, USA) was used as a loading control and subsequent quantification. The expression levels were evaluated by the AlphaView (Protein-simple, CA, USA) or Image Quant Software (Uppsala, Sweden).
RNA preparation and qRT-PCR
RNA was isolated from cell lines and EXOs using the “Total RNA Purification micro Kit” (NorgenBioteK Corp, Canada) according to the manufacturer’s protocol. In the first setting of experiment, to avoid any possible contamination of external RNAs, before exosome purification conditioned media were pre-treated with RNase (Roche, Nutley, NJ, USA) for 10 min at 37 °C. Real time quantification (qRT-PCR) of miR-222 (#000525), p27Kip1 (#Hs00153277_m1), FGF2 (#Hs00266645_m1), VEGFA (#Hs000900055_m1), ITGβ3 (#Hs00173978_m1) and Bcl-2 (#Hs00153350_m1) were performed according to the TaqMan technology (Applied Biosystems, Foster City, CA, USA). MiR-16 (#000391), RNU6B (#001093) and GAPDH (4326317E) were used as internal controls. The expression profile of Human Tumor metastasis genes was performed using the TaqMan Array 96-Well Plate (#4414229) (Applied Biosystems, Foster City, CA, USA).
Differences were statistically evaluated using Student’s t test. p < 0.05 was defined as statistically significant. ANOVA analysis was performed using GraphPad version 4.0 for Windows (GraphPad Software, San Diego, CA) followed by Student–Newman–Keuls post hoc test when appropriate.
Characterization of melanoma purified EXOs
MiR-222 overexpression is involved in the poor prognosis of several tumors where it was demonstrated to promote cell growth, migration and invasion and to inhibit apoptosis [14, 15]. In addition circulating miR-222 has been proposed as diagnostic and prognostic marker .
Melanoma-derived vesicles were then characterized for their size distribution by using the Nanosight™ technology. Results showed similar profiles for UC- and EQ-derived EXOs, with nanoparticles ranging between 70 and 140 nm, with a mean value of 100 nm, in both control and miR-222 overexpressing cells (Fig. 2b, c and data not shown). The amount of melanoma-released EXOs was also assessed by using either the Nanosight™ technology or the Bradford assay for protein quantitation. In both cases results revealed that miR-222-transduced melanomas secreted a significantly higher number of EXOs compared with control cells, possibly suggesting a role for miR-222 in the EXO releasing process (Fig. 2d).
The protein content of these EXOs analyzed by western blot showed a miR-222-dependent enrichment for proteins commonly utilized as exosomal markers, such as LAMP2, HSP90, CD63 and RAB5B, (Fig. 2e). In addition, besides confirming miR-222 overexpression into the exosomal cargo (Fig. 2a), we observed reduced levels of p27Kip1, a negative regulator of cell cycle previously demonstrated as a direct target of this miR . These results suggested that miR-222 was somehow functional in the vesicular fraction (Fig. 2e). Accordingly, in Me1402/R we detected a miR-222-dependent increase of CAV-1 and RAB27A, proteins already described for their involvement in exosome uptakes and secretory pathways [17–19]. Although CAV-1 and RAB27A are expressed in Me1007 cells, we were unable to detect them in the corresponding EXOs, likely because of their low levels. As internal loading controls, we utilized TSG101 and β-ACTIN which appeared constantly expressed (Fig. 2e).
In vitro functional studies on EXO/miR-222
To determine whether the EXO/miR-222 and their cargo could be taken up by recipient cells, we utilized an in vitro experimental model where EXOs isolated from donor cells were incubated with control melanoma cell lines. Specifically the same amounts of EXOs, purified either from miR-222- or from empty vector-transduced melanoma cells, were incubated with Me1007 or Me1402/R acceptor cells.
Above all the horizontal transfer was confirmed by specific qRT-PCR for miR-222. Indeed the uptake of miR-222-containing EXOs by the acceptor melanoma cells produced 3-fold increase of miR-222 respect to its basal expression in Me1402/R/Tween cells and 1.5-fold respect to the homologous control fusion with EXO/Tween (Fig. 3b right). Similar results were obtained in Me1007 melanoma cells (2.5-fold increase of miR-222 respect to its basal cellular expression and 1.3-fold respect to the EXO/Tween control Fig. 3b left). Of note was the downregulation of p27Kip1 consequent to EXO/miR-222 internalization comparable to that obtained by miR-222 lentiviral-induced overexpression (Fig. 3c). In addition the horizontal transfer was confirmed by changes of some other relevant molecules. As suggested by their higher expression in EXO/miR-222 respect to EXO/Tween (Fig. 2e), we observed the exosome-related capability to convey vesicle-markers possibly associated with tumorigenesis, such as CD63, CAV-1, RAB5B and RAB27A, into the acceptor cells (Additional file 1: Fig. S1).
Activation of the PI3K/AKT pathway by EXO-miR-222
To further dissect the role of miR-222 in the exosome cargo of Me1402/R melanoma, we compared the expression profiles of EXO/miR-222 and EXO/Tween by using the TaqMan array plate for human tumor metastasis genes (Additional file 3: Fig. S3A). Although current reports have shown that a fraction of the RNAs present in EXOs could be somewhat degraded, the presence of some poly-adenylated RNAs was also reported [29, 30]. As possibly expected in view of the low amounts of RNA recovered from EXOs, qRT-PCR analyses revealed that the majority of genes exhibited Ct values higher than 35. Even so, in agreement with miR-222 tumor promoting function, we observed the up-regulation of a number of genes involved in melanoma progression. Among them we found the miR-222-based upregulation of VEGF and FGF2 well known to play major roles in melanoma cell growth and tumor angiogenesis according to autocrine and paracrine functions [31, 32]. Interestingly, some other factors, as the MGAT5, MCAM and TGFβ1, cooperating in the induction of prometastatic phenotypes in melanoma [33, 34], were induced by miR-222. Finally, among the few genes downregulated in EXO/miR-222 we detected MTA1 and MTA2, nuclear receptor coregulators overexpressed in human cancers, but reported to play a dual role being either corepressors or coactivators . The accuracy of these microarray results was validated by qRT-PCR of FGF2 and VEGF genes (Additional file 3: Fig. S3B).
MiRs are non-coding RNAs regulating gene expression mostly at post-transcriptional level . Growing evidences are showing their functional involvement in all the main biological processes, including cancer development and progression, where they can act either as oncogenes or tumor suppressors . Besides their cellular association, recent data demonstrated miRs to be secreted in the extracellular compartments as free molecules or carried by microvesicles, thus transferring information at local and distant sites .
Several groups have focused their attention on miR-221 and miR-222 reported to drive the oncogenesis of many types of malignancies by directly repressing a number of tumor suppressor genes and activating oncogenic pathways . Focusing on melanoma, we previously showed that miR-221&222 play a dynamic role regulating proliferation and progression repressing several antineoplastic targets (e.g., p27Kip1/CDKN1B, c-KIT receptor and c-FOS) [6, 7].
Here we show that miR-222, being part of melanoma exosomal cargo, can be transferred between cells resulting per se able to promote tumorigenesis through the activation of several molecules, including the PI3K/AKT pathway.
Since a definite consensus on the ‘‘correct’’ technique to isolate EXOs is still lacking , we utilized and compared ultracentrifugation and Exoquick alternative methods. Purified EXOs, analyzed by NanoSight and/or western blot technologies, showed the same distribution as well as similar enrichment for some vesicular proteins, as LAMP2, CD63, RAB5B, TSG101, HSP90 and β-ACTIN (Fig. 2). In agreement with the general gradual increase of miR-222 associated with melanoma progression, either in stabilized or early passage cell lines, we detected the same expression pattern in the corresponding EXOs (Fig. 1). Moreover a significant accumulation of this miR was detectable in EXOs purified from primary melanoma cell lines (Me1007 and Me1402/R) enforced to express miR-222 (Fig. 2). Indeed the mature sequence of miR-222 contains two short sequence motifs reported to function as exosomal packaging signals or miR intracellular retaining .
Previous studies already provided evidences of exosome transfer and content deliver from donors to recipient cells [30, 39]. Utilizing an experimental model, we got higher invasive and chemotactic capabilities and, even more evident, vessel-like process formation associated with EXO/miR-222 internalization into primary melanomas, indicating increased melanoma malignancy (Figs. 3, 4) . The reverse effects obtained after internalization of EXOαmir-221&222 supported our conclusions (Fig. 5). An analogous EXO-based increase of malignancy was described for miR-10b in breast cancer [9, 40] where miR-10b was associated with the risk of relapse [41, 42].
Trying to dissect the downstream pathways regulated by miR-222, we demonstrated, either in melanoma cells or secreted EXOs, the miR-222-dependent induction of the PI3K/AKT pathway, associated with the expected downregulation of miR-222 direct target p27Kip1. As no significant differences were detected in the cell-cycle rates of miR-222-overexpressing cells treated or not with the AKT inhibitor BKM120 (Fig. 7a), we considered the capability of miR-222 to overcome the inhibition of the PI3K/AKT pathway, on one side considering the frequent constitutive activation of the MAPK axis, on the other the number of genes inhibiting proliferation, inducing apoptosis or generally playing tumor suppressor functions, known to be directly targeted by miR-222 . In addition PI3K/p85β was identified as a direct target of miR-126, that we recently demonstrated as part of a cross regulatory circuitry linking the oncomiR-221&222 with the tumor suppressors miR-126&126*. During melanoma progression, the expression levels of these two couples of miRs functionally move from miR-126&126* toward miR-221&222 under the regulation of the transcription factor AP2α . In line with their opposite roles, protein analyses confirmed that several miR-126&126* targeted genes were induced by miR-221&222, including AKT and PI3K/p85β.
According to the reported association between the number of released EXOs and tumor malignancy , miR-222 seemed also to augment the amount of secreted EXOs per cell. In agreement with their functional roles recently associated with cancer, some exosomal markers resulted upregulated by miR-222 (Fig. 2). Among them, RAB GTPases were implicated in membrane trafficking and exosome secretion in melanoma. Specifically RAB5B and RAB27 were shown to increase the release and transfer capability of the microvesicles being involved in tumor metastatization including melanoma [19, 44–46]. Also the tetraspanin CD63, a well known marker of microvesicles, was associated with prometastatic pathways  and evidenced together with Cav1 on plasma EXOs of melanoma patients . Previous studies linked Cav1 overexpression with melanoma malignancy showing that its secreted amounts loaded in the exosomal cargo were involved in cell migration [17, 48]. Indeed, recent studies supported the notion of the pro-metastatic role of CD63 through β-catenin induction and subsequent increase of ERK phosphorylation and PI3K/AKT pathway activity [47, 49, 50]. Although these increases might suggest the involvement of miR-222 in the EXO releasing process, at present no significant data directly correlate these exosomal markers with miR-222.
The expression profiles obtained by analyzing a panel of tumor metastasis genes further demonstrated the presence of higher levels of tumor promoting genes in EXO/miR-222. Among them we found MGAT5, which in melanoma plays a role during the transition from the vertical growth phase to the metastatic stage, together with its targets MCAM , and TGFβ expressed in most malignant melanomas and correlating with poor survival . Last but not least the increased levels of the growth factors VEGF and FGF2 found into the exosome cargo (Additional file 3: Fig. S3A), besides underlying the miR-222 induction of vascular-like structures, suggested the exosome-based transport to explain the unconventional leaderless secretion of FGF2 .
The growing understanding of cancer cell-derived vesicles, exosome-mediated uptake and transfer of the molecular cargos is making more realistic to easily evaluate EXOs in plasma from patients. Our results implicate miR-222, either cell-associated or exosome-transported, as a regulator of melanoma malignancy (Additional file 3: Fig. S3C), suggesting its potential validity as diagnostic and prognostic biomarker. Indeed, as miR-222 was reported to be under the positive control of the RAS/MAPK and upstream to PI3K/AKT signaling, its abrogation might represent a promising therapeutic option.
real time quantification
cyclin-dependent kinase inhibitor 1B
v-kit hardyzuckerman 4 feline sarcoma viral oncogene homolog
v-fos fbj murine osteosarcoma viral oncogene homolog
- BODIPY® FL C16:
tumor susceptibility gene 101 protein
melanoma-associated antigen MLA1
heat shock protein 90
lysosome-associated membrane glycoprotein 2
ras-related protein Rab-5B
phosphatidylinositol 3-kinase regulatory subunit beta
v-akt murine thymoma viral oncogene homolog 1
vascular endothelial growth factor a
- FGF 2:
fibroblast growth factor 2
transforming growth factor, beta-1
melanoma cell adhesion molecule
metalloproteinase inhibitor 1
B-cell cll/lymphoma 2
FF and ADF conceived the study, performed the experiments and co-wrote the article, CC purified and analyzed microvesicles, FP MB EP and MCE performed the experiments, RP performed immunofluorescence analysis, LP performed flow citofluorimeter analysis, AC supervised the project and wrote the article. All authors read and approved the final manuscript.
This work was partly supported by grants from the Italian Ministry of Health (RF-2010-2310494) and the Italian Association for Cancer Research (AIRC IG13247 to A.C.). We thank M. Sargiacomo for motivating discussion. We also thank G. Loreto for helping in figure preparation.
The authors declare that they have no competing interests.
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