human renal cancer cells express a novel membrane bound interleukin 15 that induces in response to the soluble interleukin 15 receptor α chain epithelial to mesenchymal transition

Although interleukin-15 (IL-15) is a powerful immunomodulatory 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 α (s-IL-15Rα) 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 (α-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-15Rα subunit that tunes its signaling potential competing with low concentrations of the s-IL-15Rα 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. [Cancer Res 2009;69(4):1561–9

Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

The ability of Interleukin-15 (IL-15) to activate many immune antitumor mechanisms renders the cytokine a good candidate for the therapy of solid tumors, particularly renal cell carcinoma. Although IL-15 is being currently used in clinical trials, the function of the cytokine on kidney's components has not been extensively studied; we thus investigated the role of IL-15 on normal and tumor renal epithelial cells. Herein, we analyzed the expression and the biological functions of IL-15 in normal renal proximal tubuli (RPTEC) and in their neoplastic counterparts, the renal clear cell carcinomas (RCC). This study shows that RPTEC express a functional heterotrimeric IL-15Rαβγc complex whose stimulation with physiologic concentrations of rhIL-15 is sufficient to inhibit epithelial mesenchymal transition (EMT) commitment preserving E-cadherin expression. Indeed, IL-15 is not only a survival factor for epithelial cells, but it can also preserve the renal epithelial phenotype through the γc-signaling pathway, demonstrating that the cytokine possess a wide range of action in epithelial homeostasis. In contrast, in RCC in vitro and in vivo studies reveal a defect in the expression of γc-receptor and JAK3 associated kinase, which strongly impacts IL-15 signaling. Indeed, in the absence of the γc/JAK3 couple we demonstrate the assembly of an unprecedented functional high affinity IL-15Rαβ heterodimer, that in response to physiologic concentrations of IL-15, triggers an unbalanced signal causing the down-regulation of the tumor suppressor gene E-cadherin, favoring RCC EMT process. Remarkably, the rescue of IL-15/γc-dependent signaling (STAT5), by co-transfecting γc and JAK3 in RCC, inhibits EMT reversion. In conclusion, these data highlight the central role of IL-15 and γc-receptor signaling in renal homeostasis through the control of E-cadherin expression and preservation of epithelial phenotype both in RPTEC (up-regulation) and RCC (down-regulation)

Evaluation du rôle du système IL-15/IL-15R dans la physiologie et la pathologie des cellules épithéliales rénales humaines

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions

Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105+). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression

Up-regulation of E-cadherin expression by rhIL-15 on RPTEC is dependent of the βc-dependent signaling pathway.

<p>The γc neutralization, as well as JAK3 or STAT5 inhibition, hamper the maintenance of E-cadherin surface expression induced by rhIL-15 on RPTEC without interfering on the E-cadherin down-regulation on rhIL-15-treated RCC7. Cells were pretreated with 1 µg/ml of neutralizing anti-IL2Rγ antibody mAb2842, 0.25 µM of JAK3 inhibitor (CP-690, 550, Calbiochem) or 100 µM of STAT5 inhibitor (STAT5 Inh., 573108, Calbiochem) for 1 h before adding the recombinant cytokine (10 pg/mL) for 5 days. Treatment with rhIL-15 and STAT5 inhibitor was renewed at day 3. White histograms refer to isotype-matched control. Mean fluorescence intensity values for each marker are shown in each histogram. The data are representative of 3 separate experiments performed using different RCC (RCC5, RCC8) and RPTEC batches.</p

Reestablishment of IL-2Rγ chain-dependent signal transduction pathway in RCC interferes with the rhIL-15-induced E-cadherin down-regulation.

<p>RCC7 were transiently transfected for 48 hours with vectors containing IL-2Rγ and/or JAK3 Human cDNA. <b>A</b>) Transient expression of IL-2Rγ and JAK3 was analyzed by immunoblotting in each transfected RCC. Immunoblotting for β-actin was used as a control for equal protein loading and transfer. <b>B</b>) Flow cytometry shows that 40 min rhIL-15 treatment did not induce STAT5 phosphorylation in IL-2Rγ- or JAK3-transfected RCC while rhIL-15 treatment induced STAT5 phosphorylation in co-transfected cells. <b>C</b>) After 48 h, transfected RCC were treated for an additional 48 h with 10 pg/mL of rhIL-15 before evaluating E-cadherin expression by flow cytometry. The introduction of either IL-2Rγ chain, JAK3 or both molecules do not modify E-cadherin expression on untreated rhIL-15 cells, while the E-cadherin down-regulation observed after 48 hours of rhIL-15 treatment was counterbalanced only in co-transfected cells. Mean fluorescence intensity values for each marker are shown in each histogram. One experiment representative of a total of three is shown.</p

Normal and tumoral renal epithelial cells express different IL-15R subtypes.

<p>Analysis of IL-15R and JAK3 expression was performed by RT-PCR (<b>A</b>) and immunoblotting (<b>B</b>) on primary normal (RPTEC) and tumoral (RCC5, RCC7, RCC8) epithelial cells and the ACHN cell line. Data show that RPTEC express the three chains of the IL-15R (αβγ) and JAK3 whereas γc and JAK3 proteins were not detected in RCC. Specific primers or Abs against IL-15Rα (AF247), IL-2Rβ (sc-1046), IL-2Rγ (sc-670) and JAK3 (sc-513) were used. PBL, TF1β, MCF7 and IFNγ-activated U937 cells were used as controls. Housekeeping β-actin was used as loading control.</p

Soluble IL-15, at physiologic concentration, differently controls E-cadherin expression in RCC and RPTEC.

<p>Immunofluorescence analysis (<b>A</b>) and immunoblot (<b>B</b>) show that 5 days rhIL-15 treatment (10 pg/mL) preserves membrane E-cadherin expression on primary normal epithelial cells RPTEC, whereas it induces its down-regulation on RCC7. The medium culture of RPTEC was not changed in order to induce the decrease of E-cadherin expression. Treatment with rhIL-15 was renewed at day 3. Histograms represent densitometry comparison of E-cadherin immunoblots normalized to β-actin in 3 different RCC (RCC5, RCC7, RCC8) cells and 3 RPTEC batches. * P<0.05.</p

Immunohistochemical staining for IL-2Rβ, IL-2Rγ and JAK3 in normal and neoplastic kidney specimens.

<p>Hematoxylin staining of biopsies from 2 normal samples reveals the presence of the normal kidney structures (glomerulus (Gl), distal (Dt) and proximal (Pt) tubules), while analysis of two different cancer specimens shows that the normal tissue architecture is totally lost and are replaced by tumor cells with clear cell morphology, characterized by optically clear cytoplasm and sharply outlined cell membrane. Whereas no difference on IL-2Rβ is observed between normal and tumoral tissue samples, the IL-2Rγ staining, localized to both proximal and distal tubuli in normal tissue samples, is not found in the tumor samples. A strong JAK3 staining is localized to both proximal and distal tubular cells of normal tissues, while a very faint JAK3 protein expression is detected in tumor samples. Two representative samples of a total of ten are shown for each staining. Negative control was subjected to all treatments omitting primary antibody. Scale bars, 50 µm. Staining was quantified using a morphometric TRIBVN software (Montrouge, France) and results are presented as histograms.</p