Michela Croce

Differentiation Therapy: Targeting Human Renal Cancer Stem Cells with Interleukin 15

BACKGROUND Many renal cancer patients experience disease recurrence after immunotherapy or combined treatments due to persistence of cancer stem cells (CSCs). The identification of reliable inducers of CSC differentiation may facilitate the development of efficient strategies for eliminating CSCs. We investigated whether interleukin 15 (IL-15), a regulator of kidney homeostasis, induces the differentiation of CD105-positive (CD105(+)) CSCs from human renal cancers. METHODS CD105(+) CSCs were cultured to preserve their stem cell properties and treated with recombinant human IL-15 (rhIL-15) to evaluate their ability to differentiate, to acquire sensitivity to chemotherapeutic drugs, and to form spheroids in vitro and tumors in vivo. Expression of stem cell and epithelial markers were studied by flow cytometry, immunocytochemistry, and immunoblotting. Identification of a CSC side population fraction and its sensitivity to chemotherapy drugs and expression of ATP-binding cassette (ABC) transporters and aldehyde dehydrogenase (ALDH) activities were determined by flow cytometry. Spheroid formation was determined in limiting dilution assay. Xenograft tumors were generated in severe combined immunodeficient mice (n = 12-18 mice per group). All statistical tests were two-sided. RESULTS CD105(+) CSCs treated with rhIL-15 at 10 pg/mL differentiated into cells expressing epithelial markers. rhIL-15 induced epithelial differentiation of all CD105(+) CSCs subsets and blocked CSC self-renewal (sphere-forming ability) and their tumorigenic properties in severe combined immunodeficient mice. Vinblastine and paclitaxel induced statistically significant higher levels of apoptosis in rhIL-15-differentiated epithelial cells compared with CD105(+) CSCs (mean percentage of apoptotic cells, vinblastine: 33% vs 16.5%, difference = 16.5%, 95% confidence interval = 12.25% to 20.74%, P = .0025; paclitaxel: 35% vs 11.6%, difference = 23.4%, 95% confidence interval = 22.5% to 24.24%, P = .0015). The higher sensitivity of rhIL-15-differentiated epithelial cells to chemotherapeutic drugs was associated with loss of detoxifying mechanisms such as ALDH and ABC transporter activities. CONCLUSION IL-15 directs the epithelial differentiation of renal CSCs and meets the criteria for a treatment strategy: CSC pool depletion and generation of differentiated nontumorigenic cells that are sensitive to chemotherapeutic agents.

Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15.

Background NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. Methodology/Principal Findings Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of “NK-ireg” cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34+ PB-HP. Finally, a small subset of NKp46+ HLA-G+ IL-10+ is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. Conclusions/Significance In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56+ CD16+ NKp30+ NKp44+ NKp46+ CD94+ CD69+ CCR7+) generated from specific pSTAT6+ GATA3+ precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.

Expression of the caspase-8 gene in neuroblastoma cells is regulated through an essential interferon-sensitive response element (ISRE)

Expression of the caspase-8 gene in neuroblastoma cells is regulated through an essential interferon-sensitive response element (ISRE)

Caspase-8 gene expression in neuroblastoma.

Abstract: Neuroblastoma (NB) is a solid tumor of infancy that presents a high rate of spontaneous regression, a phenomenon that likely reflects the activation of an apoptotic/differentiation program. Indeed, the level of expression of molecules involved in the regulation of apoptosis, such as p73 or survivin, is a prognostic factor in NB patients. The caspase‐8 gene (CASP8) encodes a key enzyme at the top of the apoptotic cascade. Although methylation of a putative regulatory region of the CASP8 gene reportedly inhibits its transcription in some MYCN‐amplified NB, our results indicate that the transcriptional inactivation of caspase‐8 occurs in a subset of primary NB independently of MYCN amplification or CpG methylation. In addition, the apoptotic agent fenretinide (4HPR) and interferon‐γ (IFN‐γ) induce caspase‐8 expression without modifying the methylation status of this gene. Nevertheless, the methylation level of CASP8 intragenic and promoter regions is higher in MYCN‐amplified tumors as compared to nonamplified samples. This phenomenon might reflect the existence of distinct DNA methylation errors in MYCN‐amplified and MYCN‐single copy tumors. To gain information on the mechanisms that regulate the expression of this crucial apoptotic gene, we searched for potential CASP8 regulatory regions and cloned a DNA element at the 5′ terminus of this gene that functionally acts as a promoter only in NB cell lines that express caspase‐8. The retinoic acid analogue 4HPR, IFN‐γ, and the demethylating agent 5‐aza‐cytidine activate this promoter in NB cells that lack endogenous caspase‐8, indicating that this element may regulate both constitutive and inducible CASP8 expression. These results indicate also that demethylation of the cellular genome may upregulate CASP8 through the action of trans‐acting factors. Our results provide new insights to the regulation of CASP8, a gene with an essential role in a variety of physiologic and pathologic conditions.

Immunotherapeutic applications of IL-15

IL-15 is a member of the IL-2 family of cytokines, which play a fundamental role in innate and adaptive immune responses. IL-15 has pleiotropic immune-enhancing activities, as it stimulates NK, T and NKT cell proliferation, survival and effector functions. In view of these properties, IL-15 is regarded as a good candidate for cancer immunotherapy. This possibility is reinforced by its low toxicity and efficacy in preclinical tumor models. The use of IL-15 to boost the immune response in HIV infection has also been proposed, although further studies are required to establish potential risks and benefits. Clinical trials of IL-15 have been initiated in cancer patients and in HIV vaccination and will elucidate the potential of IL-15-based immunotherapy. The purpose of this review is to provide an update on the potential applications of IL-15 in cancer immunotherapy and HIV infection.

IL-21: A Pleiotropic Cytokine with Potential Applications in Oncology

Interleukin- (IL-) 21 is a pleiotropic cytokine that regulates the activity of both innate and specific immunity. Indeed, it costimulates T and natural killer (NK) cell proliferation and function and regulates B cell survival and differentiation and the function of dendritic cells. In addition, IL-21 exerts divergent effects on different lymphoid cell leukemia and lymphomas, as it may support cell proliferation or on the contrary induce growth arrest or apoptosis of the neoplastic lymphoid cells. Several preclinical studies showed that IL-21 has antitumor activity in different tumor models, through mechanism involving the activation of NK and T or B cell responses. Moreover, IL-21s antitumor activity can be potentiated by its combination with other immune-enhancing molecules, monoclonal antibodies recognizing tumor antigens, chemotherapy, or molecular targeted agents. Clinical phase I-II studies of IL-21 in cancer patients showed immune stimulatory properties, acceptable toxicity profile, and antitumor effects in a fraction of patients. In view of its tolerability, IL-21 is also suitable for combinational therapeutic regimens with other agents. This review will summarize the biological functions of IL-21, and address its role in lymphoid malignancies and preclinical and clinical studies of cancer immunotherapy.

Different levels of control prevent interferon-γ-inducible HLA-class II expression in human neuroblastoma cells

The HLA class II expression is controlled by the transcriptional activator CIITA. The transcription of CIITA is controlled by different promoters, among which promoter-IV is inducible by IFN-γ. We analysed the regulation of HLA class II molecules by IFN-γ in a large series of human neuroblastoma cell lines. No induction of surface or intracellular HLA class II molecules and of specific mRNA was observed, in all neuroblastomas, with the exception of a nonprototypic cell line, ACN. In a large subset of neuroblastomas IFN-γ induced expression of CIITA mRNA, derived from promoter-IV, which was not methylated. In contrast, in another subset of neuroblastomas, CIITA was not inducible by IFN-γ and CIITA promoter-IV was either completely or partially methylated. Interestingly, the use of DNA demethylating agents restored CIITA gene transcriptional activation by IFN-γ, but not HLA class II expression. The defect of HLA class II was not related to alterations in RFX or NF-Y transcription factors, as suggested by EMSA or RFX gene transfection experiments. In addition, the transfection of a functional CIITA cDNA failed to induce HLA class II expression in typical neuroblastoma cells. Confocal microscopy and Western blot analysis suggested a defective nuclear translocation and/or reduced protein synthesis in CIITA-transfected NB cells. Altogether, these data point to multiple mechanisms preventing HLA class II expression in the neuroblastoma, either involving CIITA promoter-IV silencing, or acting at the CIITA post-transcriptional level.

Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Neuroblastoma is a stroma-poor (SP) aggressive pediatric cancer belonging to neuroblastic tumors, also including ganglioneuroblastoma and ganglioneuroma, two stroma-rich (SR) less aggressive tumors. Our previous gene-expression profiling analysis showed a different CXCL13 mRNA expression between SP and SR tumors. Therefore, we studied 13 SP and 13 SR tumors by reverse transcription quantitative real-time PCR (RT-qPCR) and we found that CXCR5b was more expressed in SP than in SR and CXCL13 was predominantly expressed in SR tumors. Then, we isolated neuroblastic and Schwannian stromal cells by laser capture microdissection and we found that malignant neuroblasts express CXCR5b mRNA, whereas Schwannian stromal cells express CXCL13. Immunohistochemistry confirmed that stroma expresses CXCL13 but not CXCR5. To better understand the role of CXCL13 and CXCR5 in neuroblastic tumors we studied 11 neuroblastoma cell lines and we detected a heterogeneous expression of CXCL13 and CXCR5b. Interestingly, we found that only CXCR5b splice variant was expressed in both tumors and neuroblastoma lines, whereas CXCR5a was never detected. Moreover, we found that neuroblastoma cells expressing CXCR5 receptor migrate toward a source of recombinant CXCL13. Lastly, neuroblastoma cells induced to glial cell differentiation expressed CXCL13 mRNA and protein. The chemokine released in the culture medium was able to stimulate chemotaxis of LA1–5S neuroblastoma cells. Collectively, our data suggest that CXCL13 produced by stromal cells may contribute to the generation of an environment in which the malignant neuroblasts are retained, thus limiting the possible development of metastases in patients with SR tumor. Mol Cancer Res; 9(7); 815–23. ©2011 AACR.

An interferon-sensitive response element is involved in constitutive caspase-8 gene expression in neuroblastoma cells.

We previously identified a 1.2 Kb DNA element (P‐1161/+16), 5′ to caspase‐8 exon‐1, that acts as promoter in caspase‐8‐positive, but not in caspase‐8‐negative neuroblastoma (NB) cells. The P‐1161/+16 DNA element regulates both constitutive and interferon IFN‐γ‐inducible caspase‐8 expression. Two GAS (IFN‐activated sequence, STAT‐1 binding site) and two ISRE (interferon sensitive response element, IRF binding site) were present in P‐1161/+16. Deletion studies indicated that elements essential for promoter activity in NB cells were present in a 167 bp region 5′ flanking exon‐1 (P‐151/+16), which contains an ISRE at position −32. The transcription initiation site was mapped by 5′ rapid amplification of cDNA end (RACE) at position −20 from caspase‐8 cDNA reference sequence. Disruption of the ISRE‐32 indicated that it is required for both constitutive and IFN‐γ‐inducible caspase‐8 expression. IRF‐1 and IRF‐2 transcription factors bind to the (−151/+16) DNA fragment in vitro. Chromatin immunoprecipitation (ChIP) assays showed that IRF‐1 and IRF‐2 bind to the DNA region at the 5′ of caspase‐8 gene in NB cells, which show constitutive expression but not in caspase‐8 negative cells. In these last cells, up‐regulation of caspase‐8 by IFN‐γ was associated to induction of IRF‐1 and IRF‐2 binding to caspase‐8 promoter and increased histone acetylation. Moreover, RNA interference experiments also supported the involvement of IRF‐1 and IRF‐2 in constitutive caspase‐8 expression in NB cells.

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 kidneys 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).