[Show abstract][Hide abstract] ABSTRACT: Prostate Cancer (PCa)-related deaths are mostly due to metastasization of poorly differentiated adenocarcinomas often endowed with neuroendocrine differentiation (NED) areas. The SNAI2/Slug gene is a major regulator of cell migration and tumor metastasization. We here assessed its biological significance in NED, and metastatic potential of PCa. SNAI2 expression was down-regulated in most PCa epithelia, in association with gene promoter methylation, except for cell clusters forming: a. the expansion/invasion front of high-grade PCa, b. NED areas, or c. lymph node metastasis. Knockdown of SNAI2 in PC3 cells down-regulated the expression of neural-tissue-associated adhesion molecules, Neural-Cadherin, Neural-Cadherin-2, Neuronal-Cell-Adhesion-Molecule, and of the NED marker Neuron-Specific Enolase, whereas it abolished Chromogranin-A expression. The metastasis-suppressor genes, Nm23-H1 and KISS1, were up-regulated, while the pluripotency genes SOX2, NOTCH1, CD44v6, WWTR1/TAZ and YAP1 were dramatically down-regulated. Over-expression of SNAI2 in DU145 cells substantiated its ability to regulate metastasis-suppressor, NED and pluripotency genes. In PCa and lymph node metastasis, expression of SOX2 and NOTCH1 was highly related to that of SNAI2. In conclusion, I. SNAI2 silencing in PCa may turn-off the expression of NED markers and pluripotency genes, while turning-on that of specific metastasis-suppressors, II. SNAI2 expression in selected PCa cells, by regulating their self-renewal, NED and metastatic potential, endows them with highly malignant properties. SNAI2 may thus constitute a key target for modern approaches to PCa progression.
[Show abstract][Hide abstract] ABSTRACT: Current therapies for Non-Small Cell Lung Cancer (NSCLC) still fail to significantly increase its survival rate. Here we asked whether Interleukin(IL)-27, which has revealed powerful antitumor activity and is toxicity-free in humans, is a promising therapeutic choice for NSCLC patients. IL-27's effects were tested on Adenocarcinoma (AC) and Squamous Cell Carcinoma (SCC) cell lines and xenograft models. IL-27Receptor(R) expression was assessed in lung tissues from 78 NSCLC patients. In vitro, IL-27 was ineffective on cancer cell proliferation or apoptosis, but fostered CXCL3/GROγ/MIP2β expression. In vitro and in vivo, IL-27 down-regulated stemness-related genes, namely SONIC HEDGEHOG in AC cells, and OCT4A, SOX2, NOTCH1, KLF4 along with Nestin, SNAI1/SNAIL, SNAI2/SLUG and ZEB1, in SCC cells. In vivo, IL-27 hampered both AC and SCC tumor growth in association with a prominent granulocyte- and macrophage-driven colliquative necrosis, CXCL3 production, and a reduced pluripotency- and EMT-related gene expression. Myeloablation of tumor-bearing hosts mostly abolished IL-27's antitumor effects. In clinical samples, IL-27R expression was found in AC, SCC, pre-cancerous lesions and tumor infiltrating myeloid cells, and correlated with advanced stages of disease. Our data suggest that even immunocompromised or advancer NSCLC patients may benefit from IL-27's antitumor properties based on its ability to drive myeloid cells towards antitumor activities, and down-regulate stemness- and EMT-related genes in cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Introduction: Prostate cancer (PCa) is a leading cause of cancer-related death in men worldwide. Understanding of immune-regulatory signals skewing PCa toward dormancy or progression is of crucial importance to identify novel targets of- or tools for- therapies.
The interleukin (IL)-27 has revealed potent anti-tumor activity in a variety of tumors, but its effects on PCa is still unclear. The IL-27 cytokine subunit p28, namely IL-30, has been recognized as a novel immune-regulatory molecule, but its role in cancer biology is completely unexplored.
We investigated the roles of both cytokines in PCa microenvironment and analyzed their clinical implications.
Materials and methods: IL-30 and IL-27 regulation of hPCa cell viability and expression of selected gene clusters was tested by flow cytometry and PCR array. Expression of both cytokines and their receptors were assessed by immunohistochemistry and RT-PCR on prostate and draining lymph nodes from PCa patients and correlated with clinic-pathologic data.
In vitro, IL-30 stimulated proliferation of hPCa cells and down-regulated their expression of CCL16, LIGHT, CKLF. IL-30 consistently down-regulated the tumor suppressor and androgen co-repressor CMTM3, and up-regulated chemerinR23. In the prostate and regional lymph node tissues from PCa patients, expression of IL-30 by CD68+, CD33+/CD11b+ and CD14+ leukocytes was associated with high-grade and metastatic stage of PCa.
In vitro, IL-27 inhibited proliferation and reduced the angiogenic potential of hPCa cells by down-regulating FLT1, COX-1, FGFR3 and up-regulating CXCL10 and TIMP3. In vivo, IL-27 reduced proliferation and vascularization, and promoted ischemic necrosis, of tumors developed after hPCa cell injection in nude mice. In patients’ prostate tissues, IL-27 was undetectable, while IL- 27R was expressed by normal epithelia and low grade PCa and lost by high tumor grade and stages. IL-27R was also expressed by CD11c+, CD4+ and CD8+ leukocytes infiltrating PCa and draining lymph nodes.
Conclusions: IL-30 supports PCa cell growth and its expression in PCa tissue is associated with advanced disease grade and stage. IL-30 may thus constitute a valuable target for modern therapeutic approaches to PCa progression. By contrast, IL-27 exerts direct anti-proliferative and anti-angiogenic effects on PCa cells, in addition to its immune-stimulatory effects, and may be of great value to rescue from prostatectomy patients with low grade, organ confined PCa.
Dangerous Liaisons traslating cancer biology into better patients management 56th Annual Meeting of the Italian Cancer Society, Ferrara; 09/2014
[Show abstract][Hide abstract] ABSTRACT: Introduction: Despite advances in the clinical management, lung cancer mortality has remained largely unchanged over the past three decades, underlying the need for new treatment strategies. Interleukin (IL)-27 has revealed potent anti-tumor effects in various tumor models and, importantly, freedom from toxicity in preclinical trials. We thus investigated whether IL-27 may function as anti-tumor agent in lung cancer.
Materials and methods: Non small cell lung cancer cell lines Calu6 and SK-MES were selected, on the basis of their expression of IL-27R, to assess IL-27 anti-tumor activity both in vitro and in vivo. These cell lines were injected s.c. in immune deficient mice, that were treated with hrIL-27, to investigate in vivo effects of the cytokine on tumor growth and development. Expression of IL-27/IL-27R was also assessed in surgical samples from lung cancer patients.
Results: In vitro, IL-27 treatment had no significant effects on proliferation and apoptosis of both cell lines. In SK-MES cells, typically endowed with spindle cell morphology, IL-27 significantly down-regulated expression of specific pluripotency and Epithelial-to-Mesenchimal-Transition (EMT) transcription factors. In both SK-MES and Calu-6 cell lines, IL-27 up-regulated the expression of the powerful granulocyte chemoattractant CXCL3. In SK-MES cells only, IL-27 also up-regulated IFN-g and down-regulated trombospondin-1 and laminin-a5 expression. In vivo, IL-27 reduced the growth of both Calu-6 and SK-MES tumors in association with areas of colliquative necrosis, a prominent infiltrate of granulocytes and macrophages, while a slight anti-angiogenic effect was detected particularly in SK-MES tumors. Myeloablative conditioning of tumor bearing mice greatly reduced the in vivo efficacy of IL-27, even if in the SK-MES tumors the phenotypic alterations associated with EMT down-regulation were still evident.
Finally, in patients’ lung cancer tissues, IL-27R was mainly expressed by stromal infiltrating immune cells and often by tumor cells in both adenocarcinomas and squamous cell lung cancers.
Conclusions: Our results highlight novel aspects of IL-27 anti-tumor activity such as I. the capability to orchestrate myeloid cell re-education towards anti-cancer activities and, II. down-regulate the expression of pluripotency and EMT related genes. These findings together with the evidence of IL-27R expression in lung cancer microenvironment strongly candidate this cytokine for its clinical use.
Dangerous Liaisons translating cancer biology into better patients management. 56th Annual Meeting of the Italian Cancer Society., Ferrara; 09/2014
[Show abstract][Hide abstract] ABSTRACT: Introduction: Prostate cancer (PCa)- related deaths are mostly due to metastatic diseases developed from high grade cancers often endowed with neuroendocrine differentiation (NED) areas. SNAI2/Slug is a zinc-finger transcription factor that acts as a key regulator of cell migration during both embryonic development and tumor metastatization. Its role in prostate cancer development and progression is not yet fully understood. We thus investigated SNAI2 gene expression, regulation and role in human PCa.
Materials and methods: Epithelium and stroma were microdissected from PCa specimens and then analysed by real-time RT-PCR to detect SNAI2 gene expression levels in both compartments. Immunostainings was used to localize the expression of SNAI2 protein and that of its down-stream target molecules in prostate samples. Genomic sequencing was performed to assess the involvement of epigenetic mechanisms in the SNAI2 gene expression regulation, while knockdown of SNAI2, by specific siRNA, was performed to clarify its biological role.
Results: SNAI2 expression was considerably down-regulated in most malignant epithelia, microdissected from PCa samples, in association with gene promoter methylation, with the exception of few cancer foci and cell clusters forming: I. Chromogranin-A+ neuroendocrine differentiation (NED) areas, II. the expansion/invasion front of high-grade PCa or, III. lymph-node metastasis.
SNAI2 was methylated in 22Rv1 and LNCaP cell lines and treatment with 5-aza-2’-deoxycytidine restored its expression, but not in PC3 cells. Depletion of SNAI2 mRNA by siRNA, in the latter cell line, increased expression levels of E-Cadh and Ep-CAM, while down-regulated the expression of N-Cadh and NED markers such as Chromogranin-A, Neuron-Specific-Enolase, Nr-CAM and N-Cadh 2. The pluripotency genes SOX2, OCT4A and NOTCH1 were also down-regulated, while the metastasis-suppressor genes KISS1 and Nm23-H1 were up-regulated.
In patient’s samples, the expression SNAI2 protein was related directly to that of SOX2, OCT4A and NOTCH1 and, inversely to that of Nm23-H1.
Conclusions: SNAI2 is expressed in selected PCa areas. Silencing of SNAI2, in most PCa epithelia, may turn off the expression of NED markers and pluripotency genes, while turn on that of metastasis-suppressor genes. These data by enlightening its role in shaping differentiation and metastatic potential of PCa, candidate SNAI2 as key therapeutic target to hamper PCa progression.
Dangerous Liaisons translating cancer biology into better patients management. 56th Annual Meeting of the Italian Cancer Society, Ferrara; 09/2014
[Show abstract][Hide abstract] ABSTRACT: HLA-G and HLA-E are HLA-Ib molecules with several immunoregulatory properties. Their cell surface expression can be modulated by different cytokines. Since IL-27 and IL-30 may either stimulate or regulate immune responses, we have here tested whether these cytokines may modulate HLA-G and -E expression and function on human monocytes. Monocytes expressed gp130 and WSX-1, the two chains of IL27 receptor (R), and IL6Rα (that serves as IL-30R, in combination with gp130). However, only IL27R appeared to be functional, as witnessed by IL-27 driven STAT1/ STAT3 phosphorylation. IL-27, but not IL-30, significantly upregulated HLA-E (but not HLA-G) expression on monocytes. IFN-γ; secretion by activated NK cells was dampened when the latter cells were cocultured with IL-27 pretreated autologous monocytes. Such effect was not achieved using untreated or IL-30 pretreated monocytes, thus indicating that IL-27 driven HLA-E upregulation might be involved, possibly through the interaction of this molecule with CD94/NKG2A inhibitory receptor on NK cells. In contrast, cytotoxic granules release by NK cell in response to K562 cells was unaffected in the presence of IL-27 pretreated monocytes. In conclusion, we delineated a novel immunoregulatory function of IL-27 involving HLA-E upregulation on monocytes that might in turn indirectly impair some NK cell functions.
Journal of Immunology Research 03/2014; 2014(3):938561. DOI:10.1155/2014/938561 · 2.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Different cytokines play crucial roles in inflammation and in polarizing immune responses, including IL-27 that exerts pro- and anti-inflammatory functions. Although the activity of IL-27 is well characterized in murine immune cells, only limited information is available regarding the natural cellular sources of IL-27 in humans and its effects on human immune cells. Dendritic cells (DCs) are the most potent professional APCs that in the immature state are positioned throughout peripheral tissues by acting as sentinels, sensing the presence of Ags. Activated DCs migrate into the lymph nodes and direct Ag-specific T cell responses, thus acting as key players in both adaptive and innate immunity. In this study we asked whether IL-27 is produced by human secondary lymphoid organs and what is its functional role on human DCs. To our knowledge, we provide the first evidence that 1) in lymph nodes, macrophages are the major source for IL-27; 2) immature and mature human DCs express functional IL-27R; 3) IL-27 exerts immunosuppressive activity by crippling the Ag processing machinery in immature DCs under steady-state conditions and after pulsing with a viral Ag; and 4) IL-27 is chemotactic for human DCs. Our findings highlight novel mechanisms underlying the immunosuppressive activity of IL-27, suggesting that this cytokine may function as a homeostatic cytokine in secondary lymphoid organs by limiting duration and/or intensity of ongoing adaptive immune responses. The results presented in this study pave the way to future studies aimed at investigating whether dysregulation of IL-27 expression and function may be involved in pathogenesis of autoimmune disease and cancer.
The Journal of Immunology 02/2014; 192(6). DOI:10.4049/jimmunol.1302656 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prostate cancer (PCa) is of increasing significance worldwide as a consequence of the population ageing. Fragile elderly patients may particularly benefit from noninvasive and well tolerable immunotherapeutic approaches. Preclinical studies have revealed that the immune-regulatory cytokine IL-27 may exert anti-tumor activities in a variety of tumor types without discernable toxicity. We, thus, investigated whether IL-27 may function as anti-tumor agent in human (h) PCa and analyzed the rationale for its clinical application.
In vitro, IL-27 treatment significantly inhibited proliferation and reduced the angiogenic potential of hPCa cells by down-regulating the pro-angiogenesis-related genes fms-related tyrosine kinase (FLT)1, prostaglandin G/H synthase 1/cyclooxygenase-1 (PTGS1/COX-1) and fibroblast growth factor receptor (FGFR)3. In addition, IL-27 up-regulated the anti-angiogenesis-related genes such as CXCL10 and TIMP metallopeptidase inhibitor 3 (TIMP3). In vivo, IL-27 reduced proliferation and vascularization in association with ischemic necrosis of tumors developed after PC3 or DU145 cell injection in athymic nude mice. In patients' prostate tissues, IL-27R was expressed by normal epithelia and low grade PCa and lost by high tumor grade and stages. Nevertheless, IL-27R was expressed by CD11c+, CD4+ and CD8+ leukocytes infiltrating the tumor and draining lymph nodes.
These data lead to the conclusion that i) IL-27's anti-PCa potential may be fully exploited in patients with well-differentiated, localized IL-27R positive PCa, since in this case it may act on both cancerous epithelia and the tumor microenvironment; ii) PCa patients bearing high grade and stage tumor that lack IL-27R may benefit, however, from IL-27's immune-stimulatory properties.
[Show abstract][Hide abstract] ABSTRACT: The IL-27 cytokine subunit p28, also called IL-30, has been recognized as novel immunoregulatory mediator endowed with its own functions. These are currently the subject of discussion in immunology, but completely unexplored in cancer biology. We set out to investigate IL-30's role in prostate carcinogenesis and its effects on human (h) prostate cancer (PCa) cells.
IL-30 expression, as visualized by immunohistochemistry and real-time RT-PCR on prostate and draining lymph nodes from 125 PCa patients, was correlated with clinico-pathological data. IL-30 regulation of hPCa cell viability and expression of selected gene clusters was tested by flow cytometry and PCR Array.
IL-30, absent in normal prostatic epithelia, was expressed by cancerous epithelia with Gleason ≥7 of 21.3% of PCa stage I-III and 40.9% of PCa stage IV. IL-30 expression by Tumor Infiltrating Leukocytes (T-ILK) was higher in stage IV that in stage I-III PCa (P=0.0006) or in control tissue (P=0.0011). IL-30 expression in prostate draining Lymph Nodes (LN)-ILK was higher in stage IV than in stage I-III PCa (P=0.0031) or in control nodes (P=0.0023). The main IL-30 sources were identified as CD68+macrophages, CD33+/CD11b+myeloid cells and CD14+monocytes. In vitro, IL-30 stimulated proliferation of hPCa cells and also down-regulated CCL16/LEC, TNFSF14/LIGHT, Chemokine-like-factor/CKLF and particularly CKLF-like MARVEL transmembrane-domain-containing-3/CMTM3 and greatly up-regulated ChemR23/CMKLR.
We provide the first evidence that IL-30 is implicated in PCa progression since I) its expression by PCa or T- and LN-ILK correlates with advanced disease grade and stage, and II) IL-30 exerts pro-tumor activity in hPCa cells.
Clinical Cancer Research 11/2013; 20(3). DOI:10.1158/1078-0432.CCR-13-2240 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin (IL)-18 is an immune-enhancing cytokine, which induces Interferon (IFN)-γ production, Th1-responses and anti-tumor effects. In turn, IFN-γ stimulates IL-18 Binding Protein (BP) production, which blocks IL-18 activity. In view of the potential use of IL-18 in epithelial ovarian cancer (EOC) immunotherapy, here we studied IL-18BP expression and its regulation by cytokines in EOC cells in vitro and in vivo.
Expression and production of IL-18BP in EOC cell lines, primary ovarian carcinomas and the corresponding normal tissues, patients' serum and ascites were investigated by immunochemistry, ELISA, screening of gene expression profiles and RT-PCR.
Analysis of gene expression profiles revealed that IL18BP mRNA is increased in EOC tumors compared to normal ovary cells. Release of IL-18BP was detectable in EOC sera and at greater extent in the ascites, indicating production at the tumor site. Indeed, immunochemical analyses on cells isolated from the ascites and on tumor sections indicated that IL-18BP is expressed in both tumor cells and tumor-associated leukocytes, which displayed a CD3-CD20-NKp46-CD13+CD14low phenotype. EOC cell lines do not constitutively express IL-18BP. However, its release is inducible both by IFN-γ stimulation in vitro and by xenotransplantation of EOC cells in immune-deficient mice, suggesting a role for the microenvironment. In vitro experiments and immunochemistry indicated that also IL-27 is involved in IL-18BP up-regulation in EOC cell lines and primary cells through STAT1 activation.
Altogether these data indicate that IL-18BP, which is produced in EOC in response to micro-environmental factors, may inhibit endogenous or exogenous IL-18 activity.
Clinical Cancer Research 07/2013; 19(17). DOI:10.1158/1078-0432.CCR-13-0568 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-inducible transcription factor-1 (HIF-1α) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein we explored the effect of persistent HIF-1α inhibition by a lentivirus shRNA pool on MM cell growth either in vitro or in vivo and on the transcriptional and pro-angiogenic profiles of MM cells. HIF-1α suppression did not have a significant impact on MM cell proliferation and survival in vitro although increased the anti-proliferative effect of lenalidomide. On the other hand we found that HIF-1α inhibition in MM cells down-regulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5 and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1α. The effect of HIF-1α inhibition was assessed in vivo in NOD/SCID mice both in a subcutaneous and an intratibial MM model. HIF-1α inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and VEGF immunostaining was observed. Finally, in the intra-tibial experiments HIF-1α inhibition significantly blocked bone destruction. Overall our data indicate that HIF-1α suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1α as a potential therapeutic target in MM.Leukemia accepted article preview online, 24 January 2013; doi:10.1038/leu.2013.24.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 01/2013; 27(8). DOI:10.1038/leu.2013.24 · 9.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IL-27 and TCRγδ(+) T lymphocytes play critical roles in both innate and adaptive immune responses in health and disease, including infection and tumors. Although the activity of IL-27 is well characterized in different human immune cells, no information is available on the role of IL-27 in human TCRγδ(+) T lymphocytes. Here, we provide the first evidence that TCRγδ(+) T lymphocytes express both gp130 and WSX-1 chains of IL-27R, and that IL-27 may function in TCRγδ(+) T cells by (i) inducing STAT1 and STAT3 phosphorylation, (ii) stimulating cytotoxicity against tumor cells through upregulation of cytotoxic granules production, (iii) reducing the release of Th2-related cytokines, such as IL-5 and IL-13, and inducing IFN-γ production, and (iv) upregulating the expression of CD62L. These results highlighted a novel immunoregulatory property of human IL-27 that may be relevant in the immune response against tumors. Our results may offer new perspectives for the development of future clinical trials using IL-27 and TCRγδ(+) cells for cancer immunotherapy.
European Journal of Immunology 06/2012; 42(6):1547-52. DOI:10.1002/eji.201142241 · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AML is a hematologic malignancy that represents 15-20% of all childhood acute leukemias and is responsible for more than one-half of pediatric leukemic deaths. The bulk tumor is continuously regenerated and sustained by rare leukemic ICs that proliferate slowly, thus resulting refractory to chemotherapeutic agents targeting highly proliferating cells within the tumor. Therefore, a complete eradication of the bulk tumor may depend on efficacy of therapies that target IC. In spite of the improvements in the treatment of AML, the difficulty to eradicate completely the disease incites research for innovative therapeutic approaches. In this regard, the role of cytokines in the treatment of AML has been investigated for many years, and some of them have been tested in clinical trials as a result of their immunomodulatory properties. Furthermore, recent preclinical studies highlighted the ability of the IL-12 superfamily cytokines as potent antileukemic agents that act directly on tumor cells and on leukemic IC, thus opening new perspectives for leukemic patient treatment. Here, we review the current knowledge about the antileukemic effects of cytokines, documented in preclinical and clinical studies, discussing their potential clinical application.
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia (AML) accounts for more than half of fatal cases in all pediatric leukemia patients; this observation highlights the need of more effective therapies. Thus, we investigated whether interleukin (IL)-27, an immunomodulatory cytokine, functions as an antitumor agent against pediatric AML cells.
Expression of WSX-1 and gp130 on AML cells from 16 pediatric patients was studied by flow cytometry. Modulation of leukemia cell proliferation or apoptosis upon IL-27 treatment in vitro was tested by bromodeoxyuridine/propidium iodide (PI) and Ki67, or Annexin V/PI staining and flow cytometric analysis. The angiogenic potential of AML cells treated or not with IL-27 was studied by chorioallantoic membrane assay and PCR array. In vivo studies were carried out using nonobese diabetic/severe combined immunodeficient (NOD/SCID)/Il2rg(-/-) mice injected intravenously with five pediatric AML cell samples. Leukemic cells engrafted in PBS and IL-27-treated animals were studied by immunohistochemical/morphologic analysis and by PCR array for expression angiogenic/dissemination-related genes.
We provided the first demonstration that (i) AML cells injected into NOD/SCID/Il2rg(-/-) mice gave rise to leukemia dissemination that was severely hampered by IL-27, (ii) compared with controls, leukemia cells harvested from IL-27-treated mice showed significant reduction of their angiogenic and spreading related genes, and (iii) similarly to what was observed in vivo, IL-27 reduced in vitro AML cell proliferation and modulated the expression of different genes involved in the angiogenic/spreading process.
These results provide an experimental rationale for the development of future clinical trials aimed at evaluating the toxicity and efficacy of IL-27.
Clinical Cancer Research 03/2012; 18(6):1630-40. DOI:10.1158/1078-0432.CCR-11-2432 · 8.19 Impact Factor