Human Renal Cancer Cells Express a Novel Membrane-Bound Interleukin-15 that Induces, in Response to the Soluble Interleukin-15 Receptor A Chain, Epithelial-to-Mesenchymal Transition

Although interleukin-15 (IL-15) is a powerful immunomodu-latory factor that has been proposed for cancer immunotherapy, its intratumoral expression may be correlated with tumor progression and/or poor clinical outcome. Therefore, neoplasias potentially sensitive to immunotherapy should be checked for their IL-15 expression and function before choosing immunotherapy protocols. Primary human renal cancer cells (RCC) express a novel form of membrane-bound IL-15 (mb-IL-15), which displays three major original properties: ( a ) It is expressed as a functional membrane homodimer of 27 kDa, ( b ) it is shed in the extracellular environment by the metalloproteases ADAM17 and ADAM10, and ( c ) its stimulation by soluble IL-15 receptor A (s-IL-15R A ) chain triggers a complex reverse signal (mitogen-activated protein kinases, FAK, pMLC) necessary and sufficient to ~induce epithelial-mesenchymal transdifferentiation (EMT), a crucial process in tumor progression whose induction is unprecedented for IL-15. In these cells, complete EMT is characterized by a dynamic reorganization of the cytoskeleton with the subsequent generation of a mesenchymal/contractile phenotype ( A -SMA and vimentin networks) and the loss of the epithelial markers E-cadherin and ZO-1. The retrosignaling functions are, however, hindered through an unprecedented cytokine/receptor interaction of mb-IL-15 with membrane-associated IL-15R A subunit that tunes its signaling potential competing with low concentrations of the s-IL-15R A chain. Thus, human RCC express an IL-15/IL-15R system, which displays unique biochemical and functional properties that seem to be directly involved in renal tumoral progression.


Introduction
Interleukin-15 (IL-15) is a proinflammatory cytokine that plays an important role in both the innate and adaptive immune system. IL-15 promotes the activation of neutrophils and macrophages and is critical to dendritic cell function. In addition, IL-15 is essential to the development, homeostasis, function, and survival of natural killer (NK) cells, NK T cells, and memory CD8+ T cells. Based on these properties, IL-15 has been proposed as a useful cytokine for immunotherapy (1).
IL-15 is a 14-kDa cytokine that belongs to the four-helix bundle cytokine family and was first characterized by its ability to substitute IL-2. Indeed, IL-15 shares with IL-2 the IL-2 receptor (IL-2R) h and g chains but has a unique private a chain (IL-15Ra) that is responsible for high-affinity binding and signal transduction (13). In addition, IL-15Ra may be secreted as a functional soluble molecule (s-IL-15Ra) that behaves as an agonist or antagonist for IL-15, depending on its cleavage mechanism certainly in mouse and probably in human (14)(15)(16).
Three different functional forms of IL-15 have been identified: (a) the soluble cytokine secreted by accessory cells (at pg/mL) that activates cells expressing high-affinity IL-15R (1), (b) the hyper IL-15 (s-IL-15/IL-15Ra complex) 100-fold more efficient than the noncomplexed soluble cytokine (17,18), and (c) the membranebound forms (mb-IL-15) considered as the dominant physiologic forms of the cytokine (17,19,20). Thus, a mb-IL-15 anchored through IL-15Ra, which activates by transpresentation of bystander cells, has been described. This mechanism is essential for lymphoid homeostasis, both in mice and man. In addition, human cells display two other forms of mb-IL-15. The first one, anchored at the surface of spleen stromal cells through the IL-15Rahgc complex, is able to induce NK differentiation (17). The other one is an IL-15R-independent mb-IL-15 that can function as a receptor and participate in reverse signaling and/or act in a juxtacrine fashion (19).
IL-15 has been involved in renal inflammatory pathologies in mice and human but with contrasting conclusion between the two species (21,22). Furthermore, cytosolic and nonsecreted IL-15 protein was reported in renal cell carcinoma (RCC; ref. 23), whereas we have recently reported that human RCC express a mb-IL-15 form that is able to protect NK cells from apoptosis (24).
To determine the role of IL-15 in human renal cancer progression, we here investigated the expression and function of the IL-15/IL-15R complex in RCC. Our data show that RCCs express a functional IL-15/IL-15R system characterized by unique properties apparently ''specific'' to RCCs that finely tune and shape the functions of this cytokine. Indeed, RCCs express a novel mb-IL-15 form, likely expressed as a homodimer of 27 kDa (mb-IL-15), sensitive to metalloproteases. In response to a soluble specific ligand (s-IL-15Ra), tumor IL-15 activates a reverse signal that induces epithelial-mesenchymal transdifferentiation (EMT). These signaling functions are, however, modulated through the interaction of mb-IL-15 with membrane-associated IL-15Ra subunit present in the same cell, which tunes its signaling potential.
Flow cytometry analysis. Flow cytometry cell surface analysis was performed as previously described (22). For each sample, 10,000 cells were acquired for data analysis using FACSCalibur and analyzed with CellQuest software (BD Biosciences).
Reverse transcription-PCR analysis. Total RNA was isolated by the NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer's instructions. Total RNA (1 Ag) was then subjected to the SuperScript II reverse transcriptase (Invitrogen) in 20 AL. cDNA (2 AL) was separately amplified in 25 AL, with 2.5 IU Taq polymerase (Genecraft), in the presence of 1 Amol/L of the primers specific for IL-15, IL-15Ra, or the g c , and for the housekeeping gene h-actin. The amplifications were carried out in a PCR Sprint thermal cycler (Hybaid) for 35 or 25 cycles for h-actin (15 s at 94jC, 15 s at 60jC, 30 s at 72jC) with a final extension at 72jC for 5 min. PCR products (10 AL) were then analyzed in 1% to 2% agarose gel containing ethidium bromide. Specific primers are detailed in Supplementary Table S1.
Plasma membrane and cytosolic fractions separation. Cells were washed twice with ice-cold PBS and subsequently homogenized with glass Dounce homogenizer (10 mmol/L Tris, 0.25 mmol/L sucrose, 2 mmol/L EDTA, 2 mmol/L protease inhibitors). The homogenate was first centrifuged at 2,500 Â g for 30 min at 4jC to pellet nuclei and whole cells and then at 100,000 Â g for 30 min at 4jC (cytosolic fraction). The pellet containing the plasma membrane was resuspended in 10 mmol/L Tris (pH 7.4), 150 mmol/ L NaCl, 2 mmol/L CaCl 2 , 2 mmol/L MgCl 2 , 1% digitonin, and 2 mmol/L protease inhibitors. The detergent-insoluble materials were removed by centrifugation for 15 min at 13,000 rpm at 4jC (plasma membrane fraction). Immunoprecipitations were performed on both plasma membrane and cytosolic fractions (17).
Immunoprecipitation and immunoblotting. For immunoprecipitation and Western blotting, cells were lysed in 1% digitonin cell extraction buffer [1 mmol/L EDTA, 150 mmol/L NaCl, 20 mmol/L Tris (pH 8.0), and 10% glycerol] and protease inhibitor cocktail tablets (complete, Roche). For immunoprecipitation, lysates and plasma membrane fraction were first precleared with the protein G-sepharose (GE Healthcare Bioscience AB) then incubated overnight at 4jC in 0.1% digitonin buffer with 5 Ag/mL of antibodies against IL-15 bound to protein G-sepharose or antigoat IgG bound to protein G-sepharose as control. After washing, the immunocomplexes were boiled 5 min in SDS sample buffer and analyzed in 15% SDS-PAGE. The resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon). Blots were blocked for 2 h in PBS with 0.1% Tween 20 and 5% nonfat milk. Immunoreactive proteins were detected using the Super Signal West Pico and West Fempto kits (Pierce). In some experiments, DTT was added at 20 mmol/L to PVN-treated RCC7 cell supernatant. After 20 min, at 60jC, iodoacetamide was added at 100 mmol/L and incubated in the dark for 20 min at 20jC. SDS sample buffer was subsequently added, and proteins were then separated by 15% SDS-PAGE without using any reducing agent. IL-15 was visualized by Western blot analysis.
Phosphospecific Western blotting. For signal transduction, cells were washed twice with Dulbecco's PBS and serum-starved overnight. RCCs were then stimulated with s-IL-15Ra (1-500 ng/mL) for periods ranging between 15 min and 4 h. Activation was interrupted by adding two to three volumes of ice-cold PBS. Cell pellets were lysed for 30 min on ice in 1% Triton X-100, 20 mmol/L Tris-HCl (pH 7.4), 137 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L sodium pyrophoshate, and 10% glycerol in the presence of the phosphatases inhibitors (25 mmol/L h-glycerophophate and 1 mmol/L sodium orthovanadate) and the protease inhibitor cocktail tablets. The detergent-insoluble material was removed by centrifugation at 12,000 rpm for 15 min at 4jC. The resolved proteins were transferred onto PVDF membranes and processed as described previously (27).
Induction of EMT of RCC. Exponentially growing RCC were incubated in normal growth medium containing 5 ng/mL TGF-h or 100 ng/mL of s-IL-15Ra on Lab-Tek chamber slide system 177445 (Nalge Nunc International) for 6 d. Subsequently, cells were washed twice with PBS and then fixed in 4% paraformaldehyde for 30 min on ice. Before staining with anti-a-SMA, anti-ZO-1, or anti-vimentin antibodies, RCC were permeabilized with 0.1% TX-100 for 5 min on ice, then saturated in 0.1% BSA for 30 min on ice. E-cadherin staining was performed without permeabilization. As secondary antibodies, FITC-conjugated goat anti-mouse subclasses IgG1 were used and 4 ¶,6-diamidino-2-phenylindole was added to visualize the nucleus. The cells were washed thrice with 0.1% BSA and then with PBS. The chamber slides were removed, and the samples were mounted in Mowiol by adding a glass coverslip. All samples were observed with an oil immersion objective (Plan-Neofluar 40Â/1.3, Ph3; Zeiss) through an Axiophot Zeiss microscope (Zeiss). Digital images were taken with a digital color camera (Coolview; Photonic Science Ltd.) and grabber software (ImageAccess V2.04K; Imagic Bildverarbeitung AG). Western blot analysis with E-cadherin or anti-aSMA or with anti-pMLC antibodies was performed as described above.

Results
RCC express a novel mb-IL-15 independent of IL-15R. We and others have previously shown that RCC do no secrete detectable amount of free IL-15 (23, 28) but express a mb-IL-15 not anchored through the IL-15Ra chain (24). We, here, further investigate the properties of IL-15 in RCC. In Fig. 1A, flow cytometry shows that different RCC express a mb-IL-15, whose intensity is not decreased by acidic treatment that, in contrast, causes the disappearance of mb-IL-15 in UT7 erythroleukemic cells known to transpresent IL-15 through the IL-15Ra chain (28). In addition, treatment with the phosphorylated lipase C causes a significant decrease in the membrane expression of the glycosylphosphatidylinositol (GPI)-anchored CD59 protein, whereas mb-IL-15 expression in RCC7 is not affected, suggesting that mb-IL-15 is not a GPI-linked protein (19).
Reverse transcription-PCR (RT-PCR) analysis strengthens flow cytometry data showing that RCC cell lines (HIEG and ACHN), primary cultures of healthy renal tissue, primary tumor and metastasis derived from the same patient (RCC5-N, RCC5-T, RCC5-Met), and primary tumor RCC7 cells and virus transformed epithelial embryonic kidney cells (HK2) only express the 536 transcript coding for the secretable IL-15 form (29). In addition, RCC do not express the transcript for the gc chain (Fig. 1B), whereas the IL-15Ra and h subunits are detected on their surface ( Supplementary Fig. S1).
In Fig. 1C, mb-IL-15 from RCC7 cells was analyzed by Western blotting using the L-20 antibody. Data show that RCC7 cells express a specific band at 27 kDa, whereas the ''canonical'' monomeric 14-kDa IL-15 band was only detected in lipopolysaccharide-treated human monocytes. In addition, L-20 antibody recognizes the rhIL-15 monomeric form starting from the concentrations of 0.1 to 0.5 Ag/mL, whereas the use of increasing concentrations of rhIL-15 causes the appearance of a specific 27-kDa band.
To strengthen the above data, membrane fractions from three different RCC (RCC7, HIEG, and HK2) were immunoprecipitated and blotted with the anti-IL-15 antibody L-20 (Fig. 1D). This led to the detection of a single specific band of 27 kDa in the three cell lines tested.
Overall, the data presented in Fig. 1 indicate that RCC express the IL-15 protein predominantly as a 27-kDa putative homodimeric membrane-bound form, which is anchored at the cell membrane in an IL-15R and GPI-independent manner.
RCC express a mb-IL-15 sensitive to matrix metalloproteinases. It has been reported that the shedding of mb-IL-15Ra as a soluble receptor is controlled by ADAM17 (14) and possibly by other matrix metalloproteinases (MMP), but there is no evidence that such a mechanism may concern the mb-IL-15 forms. Flow cytometric analysis in Fig. 2A shows that mb-IL-15 expressed by RCC7 is cleaved and shed after 3 hours of treatment with PVN, a potent activator of MMPs (30). The mb-IL-15 shedding induced by PVN is blocked either in the presence of a large spectrum MMP inhibitor, such as Phen, or specific competitors of ADAM17 (GW280264X) and ADAM10 (GI254023X), indicating therefore that different MMPs may control the shedding of mb-IL-15. The specificity of the effects mediated by the competitors for ADAM10 and ADAM17 was assessed by flow cytometry showing that RCC7 cells express detectable amounts of ADAM10 and ADAM17 ( Fig. 2A,  inset). Similar results were observed studying HIEG and HK2 cell lines, and the above-mentioned treatments had no toxic effects on RCC (Supplementary Fig. S2).
Subsequently, we extended the flow cytometry data, analyzing by Western blot the supernatants of RCC7 cells before and after 3 hours of PVN treatment (Fig. 2B). In concentrated supernatants of PVN-treated RCC7, a single band of f25 kDa was detected, using the anti-IL-15 L-20 antibody. This IL-15-specific band is totally (Phen) or partially (ADAM10 and ADAM17 inhibitors) inhibited in supernatants of cells treated with PVN.
IL-2 spontaneously forms noncovalent and covalent selfassociations (31); thereby, we investigated whether the IL-15 homodimers detected in rhIL-15 and putative mb-IL-15 homodimers released by PVN-treated RCC were generated through SH2 bridges, incubating both samples with iodoacetamide. Western blot analysis performed in stringent reducing and denaturing conditions shows that iodoacetamide treatment (Fig. 2C) does not modify the migratory pattern of both IL-15, suggesting that their covalent dimerization is not due to SH2 bridges. Further characterization by two-dimensional electrophoresis shows that both samples display similar charges at distributions between pH 5.2 and 5.6 without differences in their original molecular weight (Fig. 2D).
In human RCC, s-IL-15RA chain triggers mb-IL-15-dependent reverse signaling. It has been reported that a mb-IL-15 constitutively expressed by PC-3 human prostate carcinoma cells (PCC) is involved in reverse signaling activation upon stimulation with low concentrations (1 ng/mL) of s-IL-15Ra (8).
We have recently shown that the mb-IL-15 form expressed by RCC7 is functional and triggers signaling in NK cells (24). We further investigate if mb-IL-15 is able to induce reverse signaling upon stimulation with specific soluble ligands in RCC. We show a significant increase of ERK1/2 phosphorylation in RCC7 cells treated with at least 100 ng/mL of s-IL-15Ra (Fig. 3A). In addition, time course experiments with 100 ng/mL of s-IL-15Ra chain show that, in RCC7 cells, there is a rapid and significant increase of mitogen-activated protein kinase (MAPK; pERK1/2, p38, pSAPK/ JNK) and FAK phosphorylation (Fig. 3B).
mb-IL-15-dependent reverse signaling induces EMT of RCC. The coordinated activation of the ERK1/2 and FAK pathways promotes early changes in the dynamic reorganization of the cytoskeleton controlling a-SMA polymerization, focal adhesion dynamics, and down-regulation of E-cadherin protein levels in several cell systems (32,33). On the other hand, activation of p38 MAPK and/or JNK pathways results in an increased a-SMA expression and physiologic regulation of intermediate filaments (34,35).
Thus, we investigated the effects of mb-IL-15 stimulation with the s-IL-15Ra chain on the morphology and cytoskeleton reorganization of RCC. Figure 4A shows that untreated control RCC7 cells display an epithelial-like morphology, confirmed by the expression of E-cadherin and ZO-1 (two major epithelial markers) and the absence of a detectable a-SMA and vimentin networks. Remarkably, RCC7, after 6 days of treatment with 5 ng/mL of TGF-h or with 100 ng/mL of s-IL-15Ra chain, lose E-cadherin and ZO-1 expression and acquire a mesenchymal morphology associated to a cytoskeletal reorganization characterized by the formation of strong a-SMA stress fibers and a diffuse vimentin network. In addition, decreased levels of E-cadherin associated to a significant increase in a-SMA synthesis and phosphorylation of the myosin light chain (MLC; a major marker of the contractile function) were detected by Western blotting (Fig. 4B), confirming immunofluorescence analyses.
Altogether, these results show that the stimulation of mb-IL-15 with s-IL-15Ra chain triggers, in RCC, EMT, illustrating a novel powerful protumoral property of IL-15.
mb-IL-15-dependent reverse signal is hindered by a specific control mechanism. In human PC-3 PCC and human monocytes, 1 ng/mL of s-IL-15Ra chain is sufficient to induce the reverse signal (8), whereas in RCC, the minimal concentration of s-IL-15Ra is 100 ng/mL. PC-3 cells, in contrast to RCC, do not express IL-15R. Thus, in RCC, mb-IL-15 interacting with IL-15Ra, on the surface of the same cell, could compete with low concentrations of s-IL-15Ra chain for mb-IL-15 binding, preventing a reverse signal. In this context, induction of a reverse signal may require much higher concentrations of the soluble ligand.
The existence of IL-15/IL-15Ra complexes is shown by immunoprecipitation experiments. Whereas IL-15 immunoprecipitated from RCC7 cells (using the L-20 antibody) and blotted using the same antibody reveals a single specific 27-kDa band, the immunoprecipitation of IL-15 membrane, reblotted with the anti-IL-15Ra mAb 147, shows a specific band of f50 kDa (Fig. 5A). To assess if the association of IL-15Ra chain with mb-IL-15 in RCC7 was able to interfere on reverse signal activation using low concentrations of the s-IL-15Ra chain (1-10 ng/mL), we tried to dissociate the IL-15Ra chain from the cell surface by short acidic treatment (pH 4.0). IL-15Ra chain is detected at the surface of human monocytes and RCC7 cells (Fig. 5B). In monocytes, the IL-15Ra chain is strongly expressed and is resistant to acidic shock. In contrast, in RCC7 cells, the IL-15Ra chain is highly sensitive to the acid buffer and totally dissociated from the cell membrane. These data suggest that the IL-15Ra chain, depending on the cell type, may be found in different compartments, which are defined on the basis of a differential sensitivity to acidic shock. Subsequently, acidic treated and untreated RCC7 cells were stimulated 3 hours later with increasing concentrations of s-IL-15Ra (1-100 ng/mL) for 15 minutes and analyzed by Western blot for the phosphorylation of MAPK ERK1/2 (Fig. 5C). Transient removal of IL-15Ra chain resulted in increased ERK1/2 phosphorylation in response to low concentrations of s-IL-15Ra (1-10 ng/mL), whereas in untreated RCC7 cells, ERK1/2 phosphorylation required stimulation with 100 ng/mL of s-IL-15Ra. Similar results were obtained using the silencing of the IL-15Ra gene by specific small interfering RNAs ( Supplementary Fig. S3) fully supporting the initial hypothesis.

Discussion
This study reports that human RCC constitutively express a novel form of mb-IL-15, which, in response to soluble specific ligands such as s-IL-15Ra chain, triggers a complex reverse signaling leading to the development of EMT, a property that favors tumor invasiveness (36,37). In addition, RCC secrete two s-IL-15Ra isoforms of 35 and 40 kDa, creating the possibility of a continuous functional cross-talk with mb-IL-15 to modify the properties of the tumor cells themselves and/or the behavior of the infiltrated immune cells.
Human RCC constitutively express a mb-IL-15 not dependent on the expression of the trimeric IL-15R complex (13,17,18,20). Indeed, short treatment with an acidic buffer (pH 4.0) does not modify mb-IL-15 present in RCC. In addition, RCC do not express the gc chain. Therefore, mb-IL-15 expressed by RCC has the characteristics of the ''so-called'' transmembrane-anchored IL-15 constitutively expressed on the cell surface of human PCC. However, two major features distinguish RCC mb-IL-15 from the PCC/monocyte mb-IL-15 form: (a) its molecular weight is 27 kDa and (b) its shedding is a 25-kDa protein induced by enzymatic cleavage operated by ADAM10 and ADAM17 metalloproteases. Natural PCC/monocyte mb-IL-15 form and recombinant IL-15 display a molecular weight of 13.5 to 14 kDa (1,14), whereas the RCC mb-IL-15 form is detected by Western blot and immunoprecipitation as a band of 27 kDa. These results are obtained using the anti-IL-15 polyclonal IgG L-20, which does not detect in RCC lysates the traditional 13.5-kDa to 14-kDa band, whereas it recognizes this form in lipopolysaccharide-treated monocyte lysates or when using the recombinant cytokine at concentrations ranging between 0.5 and 3 Ag/mL. These data strongly suggest that the 14-kDa IL-15 form is not or too faintly expressed by RCC. Because it has been recently proposed that free IL-15 is highly prone to self-aggregation (38), similar to what was previously observed for IL-2 (31), the most likely explanation is that the 27-kDa band represents a homodimer that seems to be the predominant form of IL-15 expressed by RCC. This conclusion is also strengthened by the comparison of the biochemical properties exhibited by the ''true'' homodimers detected in rhIL-15 samples and the ''putative'' IL-15 homodimers found in RCC. Indeed, monodimensional and bidimensional electrophoretic analyses performed under stringent reducing and denaturing conditions in the presence of iodoacetamide show that the recombinant and the natural IL-15 27-kDa bands share several similarities, suggesting a covalent homodimeric nature whose self-aggregation is not due to SH2 bridges.
On the other hand, in RCC, the enzymatic shedding of mb-IL-15 homodimeric form of 25 kDa is an original property that opens interesting perspectives. Indeed, the ''shed mb-IL-15'' interacting with the natural s-IL-15Ra chain codetected in the supernatant of PMA-treated RCC forms a s-IL-15 (25 kDa)/IL-15Ra (35 kDa) complex ( Supplementary Fig. S4) that could exert either agonist or antagonist effects. Indeed, similar complexes (hyper IL-15) formed in the tumor microenvironment could acquire the competence to stimulate lymphoid cells bearing the low-affinity IL-15R (17). Alternatively, the shed homodimer could act as a decoy molecule for the s-IL-15Ra. In both situations, the intratumoral immune response would be heavily modified. In addition, because the natural s-IL-15Ra forms a complex with the shed IL-15 homodimer, we propose that it could also recognize the mb-IL-15 homodimer and deliver a reverse signal in RCC.
Similar to what was previously reported for the PCC/monocyte mb-IL-15 form, the mb-IL-15 expressed by RCC is also competent for delivering a bidirectional signal. Indeed, RCC mb-IL-15 protects from apoptosis NK cells through transpresentation mechanism (24) and, in addition, triggers reverse signaling upon stimulation with s-IL-15Ra, mediating outside-to-inside signal transduction. Thus, we observed that stimulation with the s-IL-15Ra chain induces the phosphorylation of several MAPKs (ERK1/2, p38, SAPK/JNK) and FAK. The major biological effect consequent to the reverse signal is the induction of EMT. EMT is a major biological event that, in carcinoma cells, leads to the acquisition of an increased motility and, therefore, of metastatic potential and is essentially mediated by Oncostatin M, TGF-h, and/or CTGF (39,40). EMT is a complex, highly regulated process that involves the development of migratory/contractile properties (acquisition of a mesenchymal phenotype; ref. 32) associated to the loss and/or redistribution of the epithelial markers E-cadherin and ZO-1, which are essential steps for the accomplishment of EMT and the development of tumorigenesis (41). During this process, activated ERK and FAK are upstream regulators of specific machineries that affect the formation of focal adhesions through the phosphorylation of MLC, dynamic reorganization of the actin cytoskeleton with the formation of enhanced tensile stress fibers (42,43), whereas synthesis of a-SMA and organization of the intermediate filament network seem to be under the control of the p38 and SAPK/JNK pathways (44,45). On the other hand, decreased E-cadherin expression is controlled by ERK1/2 and p38 (46), whereas downregulation of ZO-1 expression seems to be uniquely controlled by ERK1/2 activation (47). In human kidney, promotion and development of EMT is based on interactions between different inflammatory factors and resident tubular epithelial cells (48)(49)(50). Herein, we show that, in RCC, stimulation of mb-IL-15 with the soluble specific ligand s-IL-15Ra is necessary and sufficient to trigger all the above-mentioned biochemical events and to induce efficient EMT, illustrating that mb-IL-15 interactions with the s-IL-15Ra chain may lead to the development of a wide spectrum of protumoral activities, which depend on the cell system explored (8,9).
Previous papers have shown that the secretion of free IL-15 is drastically controlled at both transcriptional and posttranscriptional levels, likely for limiting the availability and possibly the effects of this powerful proinflammatory factor (1). In contrast, the existence of control mechanisms that modulate the expression/ function of mb-IL- 15 has not yet been reported. In this context, we describe a third variable that distinguishes the mb-IL-15 form expressed by RCC from that detected in PCC and human monocytes. Indeed, in former ones, it is necessary to use a 100-fold higher concentration of the s-IL-15Ra chain to trigger the reverse signal. Our data indicate that the most likely explanation is that RCC express at their surface the IL-15Ra chain, whereas PCC do not express IL-15R (8) and human monocytes do not express the acidic shock-sensitive IL-15Ra detected in RCC. In these conditions, in RCC, mb-IL-15 interacts in cis with neighboring acidic shock-sensitive IL-15Ra that lock the specific epitope on mb-IL-15, competing successfully with the soluble receptor delivered at a low concentration (1 ng/mL).
In conclusion, hijacking of the IL-15/IL-15R system for promoting tumor survival and escape from host defenses is a property displayed by several human cancer cells. However, each tumor develops strategies adapted to its specific cellular environment. Indeed, whereas bronchial and colon cancer cells secrete IL-15 that, acting through classic autocrine/paracrine loops, develop proinflammatory and proangiogenic properties (9, 51), melanoma cells display both IL-15 secretion and IL-15 nuclear localization, favoring tumor escape mechanisms (7,11). On the other hand, in head and neck carcinoma, there is a correlation between secretion of the IL-15Ra chain and poor clinical outcome. In this model, the soluble receptor, interacting with IL-15 secreted by environmental cells, triggers proinflammatory effects (44). Finally, PCC and RCC express mb-IL-15 forms competent for delivering bidirectional signaling and responsible for the induction of protumoral properties (8,24). Nevertheless, four major differences distinguish the mb-IL-15 form expressed by RCC: (a) its expression as a functional homodimer, (b) its metalloprotease-dependent shedding in the tumor microenvironment, (c) the induction of EMT, and (d) the existence of a control mechanism, represented by the interaction in cis with membrane-associated IL-15Ra chain that competes with the soluble counterparts regulating the induction of reverse signals. This particular IL-15/IL-15Ra hindering interplay could represent a conversion mechanism that favors the transpresentation functions. Alternatively, it could constitute a defense mechanism to avoid continuous activation of reverse signals by the s-IL-15Ra chain secreted in the intratumoral microenvironment by the tumor cells themselves or by other cellular sources.
We cannot exclude that the peculiar mb-IL-15/IL-15Ra interactions herewith reported in RCC may be shared by other tumor cells. However, it must be stated that renal cancers seem to be much more efficient than other tumors because they associate genetic mechanisms leading to constitutive HIF activation (52) to the secretion of immunosuppressive molecules (53) and to hijacking of the IL-15/IL-15R system, which favors tumor invasiveness.

Disclosure of Potential Conflicts of Interest
The authors state that there is no conflict of interest.