Search

The Toll-like receptor 9 (TLR9) evolved to cope with pathogens, but it is expressed in a variety of tumors for reasons that are unclear. In this study, we report that neuroblastoma (NB) cells express functional TLR9. Liposome-complexed CpG oligonucleotides inhibited the proliferation of TLR9-expressing NB cells and induced caspase-dependent apoptotic cell death. Inhibitory oligonucleotides (iODNs) abrogated these effects. RNA interference reduced TLR9 expression but not to the level where functional responses to CpG were abolished. Compared with free CpG, liposomal formulations of NB-targeted CpG (TL-CpG) significantly prolonged the survival of mice bearing NB tumor xenografts. While CpG alone lacked antitumor efficacy in NOD/SCID/IL2rg−/− mice, TL-CpG retained significant efficacy related to direct effects on tumor cells. TLR9 expression in primary human NB specimens was found to correlate inversely with disease stage. Our findings establish functional expression of TLR9 in NB and suggest that TLR9 may represent a novel theranostic target in this disease. Cancer Res; 70(23); 9816–26. ©2010 AACR.

HLA-G is overexpressed in different tumors and plays a role in immune escape. Because no information is available on HLA-G in relation to human neuroblastoma, we have investigated the expression of membrane-bound and secretion of soluble isoforms of HLA-G in neuroblastoma and functionally characterized their immunosuppressive activities. At diagnosis, serum soluble HLA-G (sHLA-G) levels were significantly higher in patients than in age-matched healthy subjects. In addition, patients who subsequently relapsed exhibited higher sHLA-G levels than those who remained in remission. Neuroblastoma patient sera selected according to high sHLA-G concentrations inhibited natural killer (NK) cell and CTL-mediated neuroblastoma cell lysis. Such lysis was partially restored by serum depletion of sHLA-G. In 6 of 12 human neuroblastoma cell lines, low HLA-G surface expression was not up-regulated by IFN-γ. Only the ACN cell line secreted constitutively sHLA-G. IFN-γ induced de novo sHLA-G secretion by LAN-5 and SHSY5Y cells and enhanced that by ACN cells. Primary tumor lesions from neuroblastoma patients tested negative for HLA-G. Neuroblastoma patients displayed a higher number of sHLA-G–secreting monocytes than healthy controls. Incubation of monocytes from normal donors with IFN-γ or pooled neuroblastoma cell line supernatants significantly increased the proportion of sHLA-G–secreting cells. In addition, tumor cell supernatants up-regulated monocyte expression of CD68, HLA-DR, CD69, and CD71 and down-regulated IL-12 production. Our conclusions are the following: (a) sHLA-G serum levels are increased in neuroblastoma patients and correlate with relapse, (b) sHLA-G is secreted by monocytes activated by tumor cells rather than by tumor cells themselves, and (c) sHLA-G dampens anti-neuroblastoma immune responses. [Cancer Res 2007;67(13):6433–41]

Previous studies have shown that the interleukin-12 receptor β2 (IL-12Rβ2) gene is expressed in normal naive, germinal center and memory B cells but not in their malignant counterparts. The aim of this study was to investigate (i) whether the IL-12Rβ2 gene is silenced in B-cell acute lymphoblastic leukemia (B-ALL) cells, and (ii) what the functional implications of such silencing for tumor growth are. Here, we show that although mature B cells expressed both chains of the IL-12R, normal pro-B and pre-B cells failed to express the IL-12Rβ2 chain. Similarly, primary tumor cells from pediatric pro-B, early pre-B, and pre-B ALL (30 cases) did not express the IL-12Rβ2 chain. IL-12Rβ2 gene silencing in B-ALL was found to depend on methylation of a CpG island in exon 1. Such methylation was not detected in normal early B cells that when differentiated into mature B cells expressed the IL-12Rβ2 gene. Detection of IL-12Rβ2 mRNA and protein in the tumorigenic 697 pre-B-ALL cell line allowed to perform functional experiments in severe combined immunodeficient/nonobese diabetic mice receiving 697 cells with or without human recombinant IL-12 (hrIL-12). hrIL-12 administration reduced tumor growth and metastasis through antiproliferative and proapoptotic rather than antiangiogenic, activities.In conclusion, epigenetic silencing of the IL-12Rβ2 gene represents a novel mechanism of tumor escape for B-ALL cells. (Cancer Res 2006; 66(8): 3978-80)

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALKhigh 12.8%, ALKlow 73%, P = 0.0035; cell death: ALKhigh 56.4%, ALKlow 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs. [Cancer Res 2009;69(18):7338–46]

Defects in the expression and/or function of the human leukocyte antigen (HLA) class I antigen-processing machinery (APM) components are found in many tumor types. These abnormalities may have a negative impact on the interactions of tumor cells with host's immune system and on the outcome of T cell–based immunotherapy. To the best of our knowledge, no information is available about APM component expression and functional characteristics in human medulloblastoma cells (Mb). Therefore, in the present study, we have initially compared the expression of APM components in Mb, an embryonal pediatric brain tumor with a poor prognosis, with that in noninfiltrating astrocytic pediatric tumors, a group of differentiated brain malignancies with favorable prognosis. LMP2, LMP7, calnexin, β2-microglobulin–free heavy chain (HC) and β2-microglobulin were down-regulated or undetectable in Mb lesions, but not in astrocytic tumors or normal fetal cerebellum. Two Mb cell lines (DAOI and D283) displayed similar but not superimposable defects in APM component expression as compared with primary tumors. To assess the functional implications of HLA class I APM component down-regulation in Mb cell lines, we tested their recognition by HLA class I antigen-restricted, tumor antigen (TA)–specific CTL, generated by stimulations with dendritic cells that had been transfected with Mb mRNA. The Mb cell lines were lysed by TA-specific CTL in a HLA-restricted manner. Thus, defective expression of HLA class I–related APM components in Mb cells does not impair their ability to present TA to TA-specific CTL. In conclusion, these results can contribute to optimize T cell–based immunotherapeutic strategies for Mb treatment. [Cancer Res 2007;67(11):5471–8]

Background/Aim The metabolic properties of cancer cells are significantly different from those of normal cells. Energy production in cancer cells is dependent on aerobic glycolysis, fatty acid synthesis and increased rates of glutamine metabolism. Emerging evidences indicate that these features are linked to therapeutic resistance in cancer treatment. Thus, novel therapeutic strategies able to modify tumor metabolism are warranted. Aim of this study is to investigate the effects and underlying mechanisms of fasting in combination with chemotherapy on tumor metabolism. Methods The in vitro citotoxicity of chemodrugs in combination with short-term-starvation (STS) was tested in various colon cancer cell lines by Trypan Blue staining and Annexin V apoptosis assay. Cancer cell proliferation by BrdUdr and CFSE staining was also evaluated. Reactive oxygen species production were evaluated by DCFDA staining. The effects of STS on the regulation of tumor metabolism were evaluated by 2-[(18)F]-Fluoro-2-deoxyglucose uptake. Expression of different proteins involved in metabolic and signaling pathways were studied by western blot and proteomic analyses. In vivo studies were performed using syngeneic BALB/c mice which were subcutaneously injected with CT26 cells. The in vivo STS protocol allowed mice to consume water but not food for 48 hours before chemotherapy. All mice were monitored daily for weight loss and side effects. Tumor growth was monitored every two days by caliper. The effects of STS in combination with chemotherapy (CT), in terms of glucose uptake/consumption and tumor growth inhibition were also evaluated by dynamic micro-Positron Emission Tomography (micro-PET). Results In vitro experiments showed that STS+CT maximally reduced cell viability and glucose uptake. A metabolic switch from anaerobic glycolysis to oxidative phosphorilation (OXPHOSPH) and beta-lipidic oxidation was induced by STS+CT. Down-regulation of PI3K/AKT pathway by STS+CT was found to be involved in the reduction of different glycolytic enzymes including hexokinases, phosphofructokinase and piruvate kinase. Furthermore, tumor cells treated with STS+CT exhibited a significant induction of respiratory chain complexes I, III, IV associated to a striking uncoupling between oxygen consumption which was induced and ATP synthesis that was reduced. This latter event was related to significant increase of ROS generation by STS+CT, leading to tumor cell apoptosis. Accordingly, in vivo experiments demonstrated that STS in combination with high-dose CT maximally reduced tumor glucose consumption in parallel with tumor cell growth. Conclusions Taken together, these results demonstrated that STS+CT affected tumor metabolism leading to a significant decrease of tumor growth. These findings open a novel scenario in cancer treatment. Citation Format: Giovanna Bianchi, Roberto Martella, Silvia Ravera, Andrea Petretto, Danilo Marimpietri, Laura Emionite, Selene Capitanio, Gianluca Bottone, Annamaria Orengo, Cecilia Marini, Gianmario Sambuceti, Vito Pistoia, Valter D. Longo, Lizzia Raffaghello. Fasting chemosensitizes tumor cells by affecting their metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3374. doi:10.1158/1538-7445.AM2014-3374