Publications by authors named "Patrizio Giacomini"

45 Publications

High activity and low toxicity of a novel CD71-targeting nanotherapeutic named The-0504 on preclinical models of several human aggressive tumors.

J Exp Clin Cancer Res 2021 Feb 10;40(1):63. Epub 2021 Feb 10.

CNR - National Research Council of Italy, Institute of Molecular Biology and Pathology, Rome, Italy.

Background: Ferritin receptor (CD71) is an example of a very attractive cancer target, since it is highly expressed in virtually all tumor types, including metastatic loci. However, this target can be considered to be inaccessible to conventional target therapies, due to its presence in many healthy tissues. Here, we describe the preclinical evaluation of a tumor proteases-activatable human ferritin (HFt)-based drug carrier (The-0504) that is able to selectively deliver the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors, preventing the limiting toxic effects associated with CD71-targeting therapies.

Methods: CD71 expression was evaluated using flow cytometry and immunohistochemistry techniques. The-0504 antiproliferative activity towards several cancer cell lines was assessed in vitro. The-0504 antitumor efficacy and survival benefit were evaluated in different human tumors, which had been grown either as xenografts or patient-derived xenografts in mice. The-0504 toxicology profile was investigated in multiple-cycle repeat-dose study in rodents.

Results: In vitro studies indicate that The-0504 is highly specific for CD71 expressing cells, and that there is a relationship between CD71 levels and The-0504 anticancer activity. In vivo treatments with The-0504 showed a remarkable efficacy, eradicating several human tumors of very diverse and aggressive histotypes, such as pancreas, liver and colorectal carcinomas, and triple-negative breast cancer.

Conclusions: Durable disease-free survival, persistent antitumor responses after discontinuation of treatment and favorable toxicology profile make The-0504 an ideal candidate for clinical development as a novel, CD71-targeted, low-toxicity alternative to chemotherapy.
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http://dx.doi.org/10.1186/s13046-021-01851-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7877078PMC
February 2021

Two distinct TP53 mutations in HNSCC primary tumor: Only one circulates in the blood.

Oral Oncol 2021 Apr 21;115:105096. Epub 2020 Nov 21.

Oncogenomic and Epigenetic Unit, IRCSS Regina Elena National Cancer Institute, Via Elio Chianesi, 53, Rome 00144, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.oraloncology.2020.105096DOI Listing
April 2021

Direct plasmonic detection of circulating RAS mutated DNA in colorectal cancer patients.

Biosens Bioelectron 2020 Dec 25;170:112648. Epub 2020 Sep 25.

Department of Chemical Sciences, University of Catania, Viale Andrea Doria, 6, 95125, Catania, Italy; INBB, Istituto Nazionale di Biostrutture e Biosistemi, Viale Delle Medaglie D'Oro, 305, 00136, Roma, Italy. Electronic address:

RAS mutations in the blood of colorectal cancer (CRC) patients are emerging as biomarkers of acquired resistance to Epidermal Growth Factor Receptor therapy. Unfortunately, reliable assays granting fast, real-time monitoring of treatment response, capable of refining retrospective, tissue-based analysis, are still needed. Recently, several methods for detecting blood RAS mutations have been proposed, generally relying on multi-step and PCR-based, time-consuming and cost-ineffective procedures. By exploiting a liquid biopsy approach, we developed an ultrasensitive nanoparticle-enhanced plasmonic method for detecting ~1 aM RAS single nucleotide variants (SNVs) in the plasma of CRC patients. The assay does not require the extraction of tumor DNA from plasma and detects it in volumes as low as 40 μL of plasma, which is at least an order of magnitude smaller than that required by state of the art liquid biopsy technologies. The most prevalent RAS mutations are detected in DNA from tumor tissue with 100% sensitivity and 83.33% specificity. Spike-in experiments in human plasma further encouraged assay application on clinical specimens. The assay was proven in plasma from CRC patients and healthy donors, and full discrimination between mutated DNA from patients over wild-type DNA from healthy volunteers was obtained thus demonstrating its promising avenue for cancer monitoring based on liquid biopsy.
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http://dx.doi.org/10.1016/j.bios.2020.112648DOI Listing
December 2020

A Distinctive microRNA (miRNA) Signature in the Blood of Colorectal Cancer (CRC) Patients at Surgery.

Cancers (Basel) 2020 Aug 25;12(9). Epub 2020 Aug 25.

Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy.

Background: Liquid biopsy (LB) provides an examination of the peripheral blood of cancer patients for circulating tumor cells, cell-free nucleic acids and microRNAs (miRNAs) and is an established tool of precision medicine. Unlike most previous LB studies that focused on advanced metastatic colorectal cancer (CRC), we assessed miRNA dysregulation in blood samples obtained on the day of surgery from patients with primary CRC lesions but no clinical evidence of extra-colonic diffusion. In this study, plasma preparation included miRNAs associated to exosomes, but excluded large macrovesicles from the preparation.

Methods: The miRNA profile in plasma isolated from a cohort of 35 CRC patients at the day of surgery was analyzed by Next Generation Sequencing (NGS) and further confirmed by droplet digital RT-PCR (dd-RT-PCR).

Results: A miR-141-3p/miR-221-3p/miR-222-3p upregulation signature previously described in advanced CRC did not discriminate the analyzed early-CRC cohort from six tumor-free donors (Tf-D). In contrast, NGS-based miRNome analysis of a training cohort of five CRC and three tumor-free donors identified a novel, distinct nine miRNA signature comprising five up-regulated and four down-regulated miRNAs, six of which could be confirmed in the full CRC and tumor-free donor validation dataset by dd-RT-PCR. Additionally, a (Kirsten Rat Sarcoma Viral Oncogene Homolog) mutant status was correlated with the plasma content of three identified miRNAs.

Conclusions: When the data obtained were comparatively evaluated, at least one of the miRNAs belonging to the signature list was found to be dysregulated in 34/35 (97.1%) of our early-CRC plasma samples. The miRNA list provides diagnostic markers as well as possible molecular targets for protocols focusing on "microRNA therapeutics".
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http://dx.doi.org/10.3390/cancers12092410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564483PMC
August 2020

Cross-sectional analysis of circulating tumor DNA in primary colorectal cancer at surgery and during post-surgery follow-up by liquid biopsy.

J Exp Clin Cancer Res 2020 Apr 20;39(1):69. Epub 2020 Apr 20.

Oncogenomics Division, Eurofins Genoma Group, Via Castel Giubileo, 11, 00138, Rome, Italy.

Background: Liquid biopsy (LB) in early-stage, non-metastatic colorectal cancer (CRC) must be sensitive enough to detect extremely low circulating tumor DNA (ctDNA) levels. This challenge has been seldom and non-systematically investigated.

Methods: Next generation sequencing (NGS) and digital PCR (dPCR) were combined to test tumor DNAs (tDNAs) and paired ctDNAs collected at surgery from 39 patients, 12 of whom were also monitored during the immediate post-surgery follow up. Patients treated for metastatic disease (n = 14) were included as controls.

Results: NGS and dPCR concordantly (100% agreement) called at least one single nucleotide variant (SNV) in 34 tDNAs, estimated differences in allelic frequencies being negligible (±1.4%). However, despite dPCR testing, SNVs were only detectable in 15/34 (44.1%) ctDNAs from patients at surgery, as opposed to 14/14 (100%) metastatic patients. This was likely due to striking differences (average 10 times, up to 500) in ctDNA levels between groups. NGS revealed blood-only SNVs, suggesting spatial heterogeneity since pre-surgery disease stages, and raising the combined NGS/dPCR sensitivity to 58.8%. ctDNA levels at surgery correlated with neither tumor size, stage, grade, or nodal status, nor with variant abundance in paired tDNA. LB sensitivity reached 63.6% when ctDNA was combined with CEA. Finally, persistence and absence of ctDNA on the first conventional (month 3) post-surgery follow-up were associated with fast relapse and a disease-free status in 3 and 7 patients, respectively.

Conclusions: A simple clinical NGS/dPCR/CEA combination effectively addresses the LB challenge in a fraction of non-metastatic CRC patients.
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http://dx.doi.org/10.1186/s13046-020-01569-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168847PMC
April 2020

Bioassay engineering: a combined label-free and fluorescence approach to optimize HER2 detection in complex biological media.

Anal Bioanal Chem 2020 May 16;412(14):3509-3517. Epub 2020 Apr 16.

Department of Basic and Applied Science for Engineering, Sapienza University of Rome, Via A. Scarpa 16, 00161, Rome, Italy.

We report on the combined label-free/fluorescence use of one-dimensional photonic crystals to optimize cancer biomarker detection in complex biological media. The optimization of the assay working parameters permits us to maximize the final response of the biosensor. The detection approach utilizes a sandwich assay, in which one-dimensional photonic crystals sustaining Bloch surface waves are modified with monoclonal antibodies in order to guarantee high specificity during biological recognition. The multiple outcomes generated by such optimization experiments permitted us to determine the effective capture efficiency and the repeatability of the immobilization process, which was estimated to be close to 5%. By exploiting the resolution of the fluorescence operation mode, we studied non-specific interactions in different blocking agents, different analyte diluting buffers, and diverse concentrations of the detection antibody. As a clinically relevant biomarker, we selected the trans-membrane receptor tyrosine kinase HER2. HER2 regulates a variety of cell proliferation, growth, and differentiation pathways and its over-expression occurs in approximately 20-30% of breast cancer worldwide. As a final application, we transferred all the optimized working parameters to HER2 cancer biomarker assays in a complex biological environment. The label-free and fluorescence results obtained by analyzing MCF-7 (HER2 low positive) and 32D (HER2 negative) cell lysates demonstrate that we can successfully discriminate the two lysates.
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http://dx.doi.org/10.1007/s00216-020-02643-3DOI Listing
May 2020

Liquid biopsy and PCR-free ultrasensitive detection systems in oncology (Review).

Int J Oncol 2018 Oct 6;53(4):1395-1434. Epub 2018 Aug 6.

Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy.

In oncology, liquid biopsy is used in the detection of next-generation analytes, such as tumor cells, cell-free nucleic acids and exosomes in peripheral blood and other body fluids from cancer patients. It is considered one of the most advanced non-invasive diagnostic systems to enable clinically relevant actions and implement precision medicine. Medical actions include, but are not limited to, early diagnosis, staging, prognosis, anticipation (lead time) and the prediction of therapy responses, as well as follow-up. Historically, the applications of liquid biopsy in cancer have focused on circulating tumor cells (CTCs). More recently, this analysis has been extended to circulating free DNA (cfDNA) and microRNAs (miRNAs or miRs) associated with cancer, with potential applications for development into multi-marker diagnostic, prognostic and therapeutic signatures. Liquid biopsies avoid some key limitations of conventional tumor tissue biopsies, including invasive tumor sampling, under-representation of tumor heterogeneity and poor description of clonal evolution during metastatic dissemination, strongly reducing the need for multiple sampling. On the other hand, this approach suffers from important drawbacks, i.e., the fragmentation of cfDNA, the instability of RNA, the low concentrations of certain analytes in body fluids and the confounding presence of normal, as well as aberrant DNAs and RNAs. For these reasons, the analysis of cfDNA has been mostly focused on mutations arising in, and pathognomonicity of, tumor DNA, while the analysis of cfRNA has been mostly focused on miRNA patterns strongly associated with neoplastic transformation/progression. This review lists some major applicative areas, briefly addresses how technology is bypassing liquid biopsy limitations, and places a particular emphasis on novel, PCR-free platforms. The ongoing collaborative efforts of major international consortia are reviewed. In addition to basic and applied research, we will consider technological transfer, including patents, patent applications and available information on clinical trials aimed at verifying the potential of liquid biopsy in cancer.
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http://dx.doi.org/10.3892/ijo.2018.4516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086621PMC
October 2018

Liquid biopsy in mice bearing colorectal carcinoma xenografts: gateways regulating the levels of circulating tumor DNA (ctDNA) and miRNA (ctmiRNA).

J Exp Clin Cancer Res 2018 Jun 26;37(1):124. Epub 2018 Jun 26.

Department of Life Sciences and Biotechnology, Biochemistry and Molecular Biology Section, Ferrara University, Via Fossato di Mortara 74, 44121, Ferrara, Italy.

Background: Circulating tumor DNA (ctDNA) and miRNA (ctmiRNA) are promising biomarkers for early tumor diagnosis, prognosis and monitoring, and to predict therapeutic response. However, a clear understanding of the fine control on their circulating levels is still lacking.

Methods: Three human colorectal carcinoma cell lines were grown in culture and as tumor xenograft models in nude mice. Chip-based and droplet digital PCR platforms were used to systematically and quantitatively assess the levels of DNAs and miRNAs released into the culture supernatants and mouse blood plasma.

Results: Strikingly, mutated DNAs from the same (KRAS) and different (PIK3CA and FBWX7) genomic loci were differentially detected in culture supernatants and blood, with LS174T releasing 25 to 60 times less DNA in culture, but giving rise to 7 to 8 times more DNA in blood than LoVo cells. Greater LS174T ctDNA accumulation occurred in spite of similar CD31 immunostaining (micro-vascularization) and lesser proliferation and tissue necrosis as compared to LoVo. As to the three selected miRNAs (miR-221, miR-222 and miR-141), all of them were constitutively present in the plasma of tumor-free mice. Micro-RNA miR-141 was released into HT-29 cell supernatants 10 and 6.5 times less abundantly with respect to LoVo and LS174T, respectively; on the contrary, release of miR-141 in blood of HT-29 xenografted mice was found similar to that observed in LoVo and LS174T mice.

Conclusions: Taken together, our results support the existence of multiple, finely tuned (non-housekeeping) control gateways that selectively regulate the release/accumulation of distinct ctDNA and miRNA species in culture and tumor xenograft models. Different xenografts (proxies of different patients) considerably differ in gateway usage, adding several layers of complexity to the well-known idea of molecular heterogeneity. We predict that even high tissue representation of mutated DNA and miRNA may result in insufficient diagnostic analyte representation in blood. In this respect, our data show that careful modeling in mice may considerably help to alleviate complexity, for instance by pre-screening for the most abundant circulating analytes in enlarged sets of tumor xenografts.
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http://dx.doi.org/10.1186/s13046-018-0788-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020232PMC
June 2018

Precision diagnostics of Ewing's sarcoma by liquid biopsy: circulating fusion transcripts.

Ther Adv Med Oncol 2018 1;10:1758835918774337. Epub 2018 Jun 1.

Oncogenomics and Epigenetics, IRCCS Regina Elena National Cancer Institute, Rome, Italy.

Background: Limited information is available on the applicative value of liquid biopsy (LB) in rare tumors, including Ewing's sarcoma (ES). The accepted precision diagnostics standards would greatly benefit from a non-invasive LB test monitoring pathognomonic gene rearrangements in the bloodstream.

Methods: Tissue and blood samples were collected from six and four ES patients, respectively. Plasma was cleared by two successive rounds of centrifugation and stored frozen until RNA extraction by the QIAmp CNA kit. RNA was retro-transcribed and subjected to real-time quantitative polymerase chain reaction (RT-qPCR) and digital polymerase chain reaction (dPCR). Reactions were set up using two custom primer sets identifying types 1 and 2 fusion transcripts.

Results: The two prevalent types of rearrangements could be identified using only two sets of polymerase chain reaction primers, regardless of patient-specific DNA breakpoints. RT-qPCR and dPCR discriminated the two variants in five tumor tissue RNAs and in four circulating tumor RNAs (ctRNAs). Of note, molecular diagnosis was possible using blood samples even when tumor tissue was not available. ctRNA levels correlated ( < 0.05) with volume-based positron emission tomography (PET) parameters (metabolic tumor volume and total lesion glycolysis), and allowed the fine tracking of disease course after surgery, during adjuvant as well as neoadjuvant chemotherapy, and at follow up in one patient.

Conclusions: To our knowledge, this is one of the few single-marker LB assays in solid tumors specifically designed to detect rearranged RNAs in blood, and the first study describing circulating tumor RNAs in ES patients. Altogether, our results support the idea that LB may have a considerable impact on ES patient monitoring and management.
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http://dx.doi.org/10.1177/1758835918774337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985603PMC
June 2018

Tearing down the walls: FDA approves next generation sequencing (NGS) assays for actionable cancer genomic aberrations.

J Exp Clin Cancer Res 2018 Mar 5;37(1):47. Epub 2018 Mar 5.

Oncogenomics and Epigenetics, IRCSS Regina Elena National Cancer Institute, Rome, Italy.

The United States Food and Drug Administration (FDA) recently approved the clinical use of two comprehensive 'mid-size' Next Generation Sequencing (NGS) panels calling actionable genomic aberrations in cancer. This is the first endorsement, by a regulatory body, of a new standard of care in oncology. Herein, we argue that besides its many practice-changing implications, this approval tears down the conceptual walls dividing system biology from clinical practice, diagnosis from research, prevention from therapy, cancer genetics from cancer genomics, and computational biology from empirical therapy assignment.
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http://dx.doi.org/10.1186/s13046-018-0702-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838869PMC
March 2018

Bloch Surface Waves Biosensors for High Sensitivity Detection of Soluble ERBB2 in a Complex Biological Environment.

Biosensors (Basel) 2017 Aug 17;7(3). Epub 2017 Aug 17.

Department of Basic and Applied Science for Engineering, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy.

We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of the cancer biomarker ERBB2 in cell lysates. Overexpression of the ERBB2 protein is associated with aggressive breast cancer subtypes. To detect soluble ERBB2, we developed an optical set-up which operates in both label-free and fluorescence modes. The detection approach makes use of a sandwich assay, in which the one-dimensional photonic crystals sustaining Bloch surface waves are modified with monoclonal antibodies, in order to guarantee high specificity during the biological recognition. We present the results of exemplary protein G based label-free assays in complex biological matrices, reaching an estimated limit of detection of 0.5 ng/mL. On-chip and chip-to-chip variability of the results is addressed too, providing repeatability rates. Moreover, results on fluorescence operation demonstrate the capability to perform high sensitive cancer biomarker assays reaching a resolution of 0.6 ng/mL, without protein G assistance. The resolution obtained in both modes meets international guidelines and recommendations (15 ng/mL) for ERBB2 quantification assays, providing an alternative tool to phenotype and diagnose molecular cancer subtypes.
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http://dx.doi.org/10.3390/bios7030033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618039PMC
August 2017

Detection of soluble ERBB2 in breast cancer cell lysates using a combined label-free/fluorescence platform based on Bloch surface waves.

Biosens Bioelectron 2017 Jun 9;92:125-130. Epub 2017 Feb 9.

Department of Basic and Applied Science for Engineering, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy.

We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of ERBB2/neu/Her2 in cell lysates. ERBB2 is a pivotal breast cancer biomarker and targetable oncogenic driver associated with aggressive breast cancer subtypes. To quantitate soluble ERBB2, we developed an optical platform that combines label-free and fluorescence detection modes. Such platform makes use of a sandwich assay in which the one-dimensional photonic crystals sustaining Bloch surface waves are tailored with a monoclonal antibody for highly specific biological recognition (BSW biochip). In a second step, a second antibody to ERBB2 quantitatively detects the bound analyte. The strategy of the present approach takes advantage of the combination of label-free and fluorescence techniques, making bio-recognition more robust and sensitive. In the fluorescence operation mode, the platform can attain the limit of detection 0.3ng/mL (1.5pM) for ERBB2 in cell lysates. Such resolution meets the international guidelines and recommendations (15ng/mL) for diagnostic ERBB2 assays that in the future may help to more precisely assign therapies counteracting cancer cell proliferation and metastatic spread.
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http://dx.doi.org/10.1016/j.bios.2017.02.012DOI Listing
June 2017

Selective delivery of doxorubicin by novel stimuli-sensitive nano-ferritins overcomes tumor refractoriness.

J Control Release 2016 10 12;239:10-8. Epub 2016 Aug 12.

Institute of Molecular Biology and Pathology, CNR - National Research Council of Italy, 00185 Rome, Italy. Electronic address:

Human ferritin heavy chain (HFt) has been demonstrated to possess considerable potential for targeted delivery of drugs and diagnostic agents to cancer cells. Here, we report the development of a novel HFt-based genetic construct (HFt-MP-PAS) containing a short peptide linker (MP) between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). The peptide linker contains a matrix-metalloproteinases (MMPs) cleavage site that permits the protective PAS shield to be removed by tumor-driven proteolytic cleavage within the tumor microenvironment. For the first time HFt-MP-PAS ability to deliver doxorubicin to cancer cells, subcellular localization, and therapeutic efficacy on a xenogeneic mouse model of a highly refractory to conventional chemotherapeutics type of cancer were evaluated. HFt-MP-PAS-DOXO performance was compared with the novel albumin-based drug delivery system INNO-206, currently in phase III clinical trials. The results of this work provide solid evidence indicating that the stimuli-sensitive, long-circulating HFt-MP-PAS nanocarriers described herein have the potential to be exploited in cancer therapy.
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http://dx.doi.org/10.1016/j.jconrel.2016.08.010DOI Listing
October 2016

Improved Doxorubicin Encapsulation and Pharmacokinetics of Ferritin-Fusion Protein Nanocarriers Bearing Proline, Serine, and Alanine Elements.

Biomacromolecules 2016 Feb 30;17(2):514-22. Epub 2015 Dec 30.

Institute of Molecular Biology and Pathology CNR, National Research Council of Italy , 00185 Rome, Italy.

A novel human ferritin-based nanocarrier, composed of 24 modified monomers able to auto-assemble into a modified protein cage, was produced and used as selective carrier of anti-tumor payloads. Each modified monomer derives from the genetic fusion of two distinct modules, namely the heavy chain of human ferritin (HFt) and a stabilizing/protective PAS polypeptide sequence rich in proline (P), serine (S), and alanine (A) residues. Two genetically fused protein constructs containing PAS polymers with 40- and 75-residue lengths, respectively, were compared. They were produced and purified as recombinant proteins in Escherichia coli at high yields. Both preparations were highly soluble and stable in vitro as well as in mouse plasma. Size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy results indicated that PASylated ferritins are fully assembled and highly monodispersed. In addition, yields and stability of encapsulated doxorubicin were significantly better for both HFt-PAS proteins than for wild-type HFt. Importantly, PAS sequences considerably prolonged the half-life of HFt in the mouse bloodstream. Finally, our doxorubicin-loaded nanocages preserved the pharmacological activity of the drug. Taken together, these results indicate that both of the developed HFt-PAS fusion proteins are promising nanocarriers for future applications in cancer therapy.
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http://dx.doi.org/10.1021/acs.biomac.5b01446DOI Listing
February 2016

Sub-apoptotic dosages of pro-oxidant vitamin cocktails sensitize human melanoma cells to NK cell lysis.

Oncotarget 2015 Oct;6(31):31039-49

Laboratory of Immunology, Regina Elena National Cancer Institute, 00144 Rome, Italy.

Alpha-tocopheryl succinate (αTOS), vitamin K3 (VK3) and vitamin C (ascorbic acid, AA) were previously shown to synergistically promote different death pathways in carcinoma cells, depending on their concentrations and combinations. Similar effects were observed herein in melanoma cells, although αTOS behaved as an antagonist. Interestingly, suboptimal cell death-inducing concentrations (1.5 μM αTOS/20 μM AA/0.2 μM VK3) effectively up-regulated activating Natural Killer (NK) cell ligands, including MICA (the stress-signaling ligand of the NKG2D receptor), and/or the ligands of at least one of the natural cytotoxicity receptors (NKp30, NKp44 and NKp46) in 5/6 melanoma cell lines. Only an isolated MICA down-regulation was seen. HLA class I, HLA class II, ULBP1, ULBP2, ULBP3, Nectin-2, and PVR displayed little, if any, change in expression. Ligand up-regulation resulted in improved lysis by polyclonal NK cells armed with the corresponding activating receptors. These results provide the first evidence for concerted induction of cell death by cell-autonomous and extrinsic (immune) mechanisms. Alarming the immune system much below the cell damage threshold may have evolved as a sensitive readout of neoplastic transformation and oxidative stress. Cocktails of vitamin analogues at slightly supra-physiological dosages may find application as mild complements of melanoma treatment, and in chemoprevention.
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http://dx.doi.org/10.18632/oncotarget.5024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741587PMC
October 2015

Up-regulation of activating and inhibitory NKG2 receptors in allogeneic and autologous hematopoietic stem cell grafts.

J Exp Clin Cancer Res 2015 Sep 11;34:98. Epub 2015 Sep 11.

Laboratory of Immunology, Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Background: Hematopoietic Stem Cell Transplantation (HSCT) is known to induce the inhibitory immune receptor NKG2A on NK cells of donor origin. This occurs in allogeneic recipients, in both the haploidentical and HLA-matched settings.

Methods: To gain further insight, not only NKG2A, but also the activating receptors NKG2C and NKG2D were assessed by flow cytometry. Immunophenotyping was carried out not only on CD56(+) but also on CD8(+) lymphocytes from leukemia and lymphoma patients, receiving both HLA-matched (n = 7) and autologous (n = 5) HSCT grafts. Moreover, cognate NKG2 ligands (HLA-E, MICA, ULBP-1, ULBP-2 and ULBP-3) were assessed by immunohistochemistry in diagnostic biopsies from three autotransplanted patients, and at relapse in one case.

Results: All the NKG2 receptors were simultaneously up-regulated in all the allotransplanted patients on CD8(+) and/or CD56(+) cells between 30 and 90 days post-transplant, coinciding with, or following, allogeneic engraftment. Up-regulation was of lesser entity and restricted to CD8(+) cells in the autotransplantation setting. The phenotypic expression ratio between activating and inhibitory NKG2 receptors was remarkably similar in all the patients, except two outliers (a long survivor and a short survivor) who surprisingly displayed a similar NKG2 activation immunophenotype. Tumor expression of 2 to 3 out of the 5 tested NKG2 ligands was observed in 3/3 diagnostic biopsies, and 3 ligands were up-regulated post-transplant in a patient.

Conclusions: Altogether, these results are consistent with a dual (activation-inhibition) NK cell re-education mode, an innate-like T cell re-tuning, and a ligand:receptor interplay between the tumor and the immune system following HSCT including, most interestingly, the up-regulation of several activating NKG2 ligands. Turning the immune receptor balance toward activation on both T and NK cells of donor origin may complement ex vivo NK cell expansion/activation strategies in unmanipulated patients.
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http://dx.doi.org/10.1186/s13046-015-0213-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4567793PMC
September 2015

Monoclonal antibodies to HLA-E bind epitopes carried by unfolded β2 m-free heavy chains.

Eur J Immunol 2015 Aug 5;45(8):2356-64. Epub 2015 Jun 5.

Laboratory of Immunology, Regina Elena National Cancer Institute, Rome, Italy.

Since HLA-E heavy chains accumulate free of their light β2 -microglobulin (β2 m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both β2 m-associated and β2 m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β2 m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4-6 residues and are "hidden" in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies.
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http://dx.doi.org/10.1002/eji.201545446DOI Listing
August 2015

Antibody-drug conjugates: targeting melanoma with cisplatin encapsulated in protein-cage nanoparticles based on human ferritin.

Nanoscale 2013 Dec;5(24):12278-85

CNR - National Research Council of Italy, Institute of Molecular Biology and Pathology, Rome, Italy.

A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4(+) melanoma cell line, but not to a CSPG4(-) breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt-Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt-Pt-Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt-Pt-Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt-Pt-Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.
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http://dx.doi.org/10.1039/c3nr04268eDOI Listing
December 2013

Comment on "Influence of HLA-C expression level on HIV control".

Science 2013 Sep;341(6151):1175

Istituto Nazionale Tumori Regina Elena, Via delle Messi d'Oro 156, 00158 Roma, Italy.

Apps et al. (Reports, 5 April 2013, p. 87) found that high human leukocyte antigen C (HLA-C) expression favors HIV-1 control. However, as noted here, HLA-C was assessed with a monoclonal antibody (DT9) that cross-reacts with HLA-E. In the context of the available evidence, this is consistent with the idea that the two leukocyte antigens collaborate to keep the HIV-1 virus at bay.
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http://dx.doi.org/10.1126/science.1241266DOI Listing
September 2013

A melanoma immune response signature including Human Leukocyte Antigen-E.

Pigment Cell Melanoma Res 2014 Jan 9;27(1):103-12. Epub 2013 Oct 9.

Laboratory of Immunology, Regina Elena National Cancer Institute, Rome, Italy.

Paired cultures of early-passage melanoma cells and melanocytes were established from metastatic lesions and the uninvolved skin of five patients. In this stringent autologous setting, cDNA profiling was used to analyze a subset of 1477 genes selected by the Gene Ontology term 'immune response'. Human Leukocyte Antigen E (HLA-E) was ranked 19th among melanoma-overexpressed genes and was embedded in a transformation signature including its preferred peptide ligand donors HLA-A, HLA-B, HLA-C, and HLA-G. Mostly undetectable in normal skin and 39 nevi (including rare and atypical lesions), HLA-E was detected by immunohistochemistry in 17/30 (57%) and 32/48 (67%) primary and metastatic lesions, respectively. Accordingly, surface HLA-E was higher on melanoma cells than on melanocytes and protected the former (6/6 cell lines) from lysis by natural killer (NK) cells, functionally counteracting co-expressed triggering ligands. Although lacking HLA-E, melanocytes (4/4 cultures) were nevertheless (and surprisingly) fully protected from NK cell lysis.
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http://dx.doi.org/10.1111/pcmr.12164DOI Listing
January 2014

Lysis-on-chip of single target cells following forced interaction with CTLs or NK cells on a dielectrophoresis-based array.

J Immunol 2013 Oct 4;191(7):3545-52. Epub 2013 Sep 4.

Silicon Biosystems, 40129 Bologna, Italy;

Guiding the interaction of single cells acting as partners in heterotypic interactions (e.g., effectors and targets of immune lysis) and monitoring the outcome of these interactions are regarded as crucial biomedical achievements. In this study, taking advantage of a dielectrophoresis (DEP)-based Laboratory-on-a-chip platform (the DEPArray), we show that it is possible to generate closed DEP cages entrapping CTLs and NK cells as either single cells or clusters; reversibly immobilize a single virus-presenting or tumor cell within the chip at a selected position; move cages and their content to predetermined spatial coordinates by software-guided routing; force a cytotoxic effector to physically interact with a putative target within a secluded area by merging their respective cages; generate cages containing effector and target cells at predetermined E:T ratios; accurately assess cytotoxicity by real-time quantitation of the release kinetics of the fluorescent dye calcein from target cells (>50 lytic events may be tested simultaneously); estimate end points of calcein release within 16 min of initial E:T cell contact; simultaneously deliver Ab-based phenotyping and on-chip lysis assessment; and identify lytic and nonlytic E:T combinations and discriminate nonlytic effector phenotypes from target refractoriness to immune lysis. The proof of principle is provided that DEPArray technology, previously used to levitate and move single cells, can be used to identify highly lytic antiviral CTLs and tumor cells that are particularly refractory to NK cell lysis. These findings are of primary interest in targeted immunotherapy.
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http://dx.doi.org/10.4049/jimmunol.1300890DOI Listing
October 2013

Programmable interactions of functionalized single bioparticles in a dielectrophoresis-based microarray chip.

Anal Chem 2013 Sep 22;85(17):8219-24. Epub 2013 Aug 22.

Silicon Biosystems, Bologna, Italy.

Manipulating single biological objects is a major unmet challenge of biomedicine. Herein, we describe a lab-on-a-chip platform based on dielectrophoresis (DEP). The DEParray is a prototypal version consisting of 320 × 320 arrayed electrodes generating >10,000 spherical DEP cages. It allows the capture and software-guided movement to predetermined spatial coordinates of single biological objects. With the DEParray we demonstrate (a) forced interaction between a single, preselected target cell and a programmable number of either microspheres or natural killer (NK) cells, (b) on-chip immunophenotypic discrimination of individual cells based on differential rosetting with microspheres functionalized with monoclonal antibodies to an inhibitory NK cell ligand (HLA-G), (c) on-chip, real-time (few minutes) assessment of immune lysis by either visual inspection or semiautomated, time-lapse reading of a fluorescent dye released from NK cell-sensitive targets, and (d) manipulation and immunophenotyping with limiting amounts (about 500) cells. To our knowledge, this is the first report describing a DEP-based lab-on-a-chip platform for the quick, arrayed, software-guided binding of individually moved biological objects, the targeting of single cells with microspheres, and the real-time characterization of immunophenotypes. The DEParray candidates as a discovery tool for novel cell:cell interactions with no prior (immuno)phenotypic knowledge.
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http://dx.doi.org/10.1021/ac401296mDOI Listing
September 2013

T and NK cells: two sides of tumor immunoevasion.

J Transl Med 2013 Feb 4;11:30. Epub 2013 Feb 4.

Paediatric Haematology/Oncology Department, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant’Onofrio 4, Rome 00165, Italy.

Natural Killer (NK) cells are known to reject several experimental murine tumors, but their antineoplastic activity in humans is not generally agreed upon, as exemplified by an interesting correspondence recently appeared in Cancer Research. In the present commentary, we join the discussion and bring to the attention of the readers of the Journal of Translational Medicine a set of recent, related reports. These studies demonstrate that effectors of the adaptive and innate immunity need to actively cooperate in order to reject tumors and, conversely, tumors protect themselves by dampening both T and NK cell responses. The recently reported ability of indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) expressed by melanoma cells to down-regulate activating NK receptors is yet another piece of evidence supporting combined and highly effective T/NK cell disabling. Major Histocompatibility Complex class I (MHC-I) molecules, including Human Leukocyte Antigen E (HLA-E), represent another class of shared activating/inhibitory ligands. Ongoing clinical trials with small molecules interfering with IDO and PGE2 may be exploiting an immune bonus to control cancer. Conversely, failure to simultaneously engage effectors of both the innate and the adaptive immunity may contribute to explain the limited clinical efficacy of T cell-only vaccination trials. Shared (T/NK cells) natural immunosuppressants and activating/inhibitory ligands expressed by tumor cells may provide mechanistic insight into impaired gathering and function of immune effectors at the tumor site.
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http://dx.doi.org/10.1186/1479-5876-11-30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3621684PMC
February 2013

IRF1 and NF-kB restore MHC class I-restricted tumor antigen processing and presentation to cytotoxic T cells in aggressive neuroblastoma.

PLoS One 2012 5;7(10):e46928. Epub 2012 Oct 5.

Paediatric Haematology/Oncology Department, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Neuroblastoma (NB), the most common solid extracranial cancer of childhood, displays a remarkable low expression of Major Histocompatibility Complex class I (MHC-I) and Antigen Processing Machinery (APM) molecules, including Endoplasmic Reticulum (ER) Aminopeptidases, and poorly presents tumor antigens to Cytotoxic T Lymphocytes (CTL). We have previously shown that this is due to low expression of the transcription factor NF-kB p65. Herein, we show that not only NF-kB p65, but also the Interferon Regulatory Factor 1 (IRF1) and certain APM components are low in a subset of NB cell lines with aggressive features. Whereas single transfection with either IRF1, or NF-kB p65 is ineffective, co-transfection results in strong synergy and substantial reversion of the MHC-I/APM-low phenotype in all NB cell lines tested. Accordingly, linked immunohistochemistry expression patterns between nuclear IRF1 and p65 on the one hand, and MHC-I on the other hand, were observed in vivo. Absence and presence of the three molecules neatly segregated between high-grade and low-grade NB, respectively. Finally, APM reconstitution by double IRF1/p65 transfection rendered a NB cell line susceptible to killing by anti MAGE-A3 CTLs, lytic efficiency comparable to those seen upon IFN-γ treatment. This is the first demonstration that a complex immune escape phenotype can be rescued by reconstitution of a limited number of master regulatory genes. These findings provide molecular insight into defective MHC-I expression in NB cells and provide the rational for T cell-based immunotherapy in NB variants refractory to conventional therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046928PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465322PMC
April 2013

Melanoma molecular classes and prognosis in the postgenomic era.

Lancet Oncol 2012 May;13(5):e205-11

Laboratory of Immunology, Regina Elena National Cancer Institute, Rome, Italy.

Gene expression profiling is a powerful method to classify human tumours on the basis of biological aggressiveness, response to therapy, and outcome for the patient, but its application in melanoma lags behind that of other cancers. From more than 100 articles available on the topic, we selected 14 focusing on patients' outcome. We review and briefly discuss salient findings, and list ten reasons why melanoma molecular classes are not yet used in clinical diagnosis and prognosis. The available evidence suggests that we are on the verge of creating a framework for the use of melanoma molecular classes in prognosis, but so far there is little consensus to put together informative diagnostic and prognostic gene sets.
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http://dx.doi.org/10.1016/S1470-2045(12)70003-7DOI Listing
May 2012

High expression of HLA-E in colorectal carcinoma is associated with a favorable prognosis.

J Transl Med 2011 Oct 27;9:184. Epub 2011 Oct 27.

Department of Pathology, Regina Elena National Cancer Institute, Via E. Chianesi 53, 00144 Rome, Italy.

Background: Human Leukocyte Antigen (HLA)-E is a non-classical class I HLA molecule that can be stabilized by ligands donated by other classical (HLA-A, -B, -C) and non-classical (HLA-G) family members. HLA-E engages a variety of immune receptors expressed by cytotoxic T lymphocytes (CTLs), Natural killer (NK) cells and NK-CTLs. In view of the opposing outcomes (activation or inhibition) of the different HLA-E receptors, the preferred role (if any) of HLA-E expressed in vivo on tumor cells remains to be established.

Methods: Taking advantage of MEM-E/02, a recently characterized antibody to denatured HLA-E molecules, HLA-E expression was assessed by immunohistochemistry on an archival collection (formalin-fixed paraffin-embedded) of 149 colorectal primary carcinoma lesions paired with their morphologically normal mucosae. Lymphoid infiltrates were assessed for the expression of the HLA-E-specific, inhibitory, non-rearranging receptor NKG2A.

Results: High HLA-E expression did not significantly correlate with the expression of classical HLA-B and HLA-C molecules, but it did correlate with high expression of its preferential ligand donor HLA-A. In addition, it correlated with lymphoid cell infiltrates expressing the inhibitory NKG2A receptor, and was an independent predictor of good prognosis, particularly in a subset of patients whose tumors express HLA-A levels resembling those of their paired normal counterparts (HLA-A). Thus, combination phenotypes (HLA-Elo-int/HLA-AE and HLA-Ehi/HLA-AE) of classical and non-classical class I HLA molecules mark two graded levels of good prognosis.

Conclusions: These results suggest that HLA-E favors activating immune responses to colorectal carcinoma. They also provide evidence in humans that tumor cells entertain extensive negotiation with the immune system until a compromise between recognition and escape is reached. It is implied that this process occurs stepwise, as predicted by the widely accepted 'immunoediting' model.
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http://dx.doi.org/10.1186/1479-5876-9-184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219584PMC
October 2011

Human leukocyte antigen E contributes to protect tumor cells from lysis by natural killer cells.

Neoplasia 2011 Sep;13(9):822-30

Laboratory of Immunology, Regina Elena Cancer Institute CRS, Rome, Italy.

The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182274PMC
http://dx.doi.org/10.1593/neo.101684DOI Listing
September 2011

Natural killer cells efficiently reject lymphoma silenced for the endoplasmic reticulum aminopeptidase associated with antigen processing.

Cancer Res 2011 Mar 20;71(5):1597-606. Epub 2011 Jan 20.

Oncohaematology Department, IRCCS, Ospedale Pediatrico Bambino Gesù, Rome, Italy.

The endoplasmic reticulum aminopeptidase ERAAP is involved in the final trimming of peptides for presentation by MHC class I (MHC-I) molecules. Herein, we show that ERAAP silencing results in MHC-I peptide-loading defects eliciting rejection of the murine T-cell lymphoma RMA in syngeneic mice. Although CD4 and CD8 T cells are also involved, rejection is mainly due to an immediate natural killer (NK) cell response and depends on the MHC-I-peptide repertoire because replacement of endogenous peptides with correctly trimmed, high-affinity peptides is sufficient to restore an NK-protective effect of MHC-I molecules through the Ly49C/I NK inhibitory receptors. At the crossroad between innate and adaptive immunity, ERAAP is therefore unique in its two-tiered ability to control tumor immunogenicity. Because a large fraction of human tumors express high levels of the homologous ERAP1 and/or ERAP2, the present findings highlight a convenient, novel target for cancer immunotherapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-3326DOI Listing
March 2011

NF-kappaB, and not MYCN, regulates MHC class I and endoplasmic reticulum aminopeptidases in human neuroblastoma cells.

Cancer Res 2010 Feb 26;70(3):916-24. Epub 2010 Jan 26.

Research Center, Ospedale Bambino Gesù, Rome, Italy.

Neuroblastoma (NB) is the most common solid extracranial cancer of childhood. Amplification and overexpression of the MYCN oncogene characterize the most aggressive forms and are believed to severely downregulate MHC class I molecules by transcriptional inhibition of the p50 NF-kappaB subunit. In this study, we found that in human NB cell lines, high MYCN expression is not responsible for low MHC class I expression because neither transfection-mediated overexpression nor small interfering RNA suppression of MYCN affects MHC class I and p50 levels. Furthermore, we identified NF-kappaB as the immediate upstream regulator of MHC class I because the p65 NF-kappaB subunit binds MHC class I promoter in chromatin immunoprecipitation experiments, and MHC class I expression is enhanced by p65 transfection and reduced by (a) the chemical NF-kappaB inhibitor sulfasalazine, (b) a dominant-negative IKBalpha gene, and (c) p65 silencing. Moreover, we showed that the endoplasmic reticulum aminopeptidases ERAP1 and ERAP2, which generate MHC class I binding peptides, are regulated by NF-kappaB, contain functional NF-kappaB-binding elements in their promoters, and mimic MHC class I molecules in the expression pattern. Consistent with these findings, nuclear p65 was detected in NB cells that express MHC class I molecules in human NB specimens. Thus, the coordinated downregulation of MHC class I, ERAP1, and ERAP2 in aggressive NB cells is attributable to a low transcriptional availability of NF-kappaB, possibly due to an unknown suppressor other than MYCN.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-2582DOI Listing
February 2010

HLA-E and the origin of immunogenic self HLA epitopes.

Mol Immunol 2010 May 22;47(9):1661-2; author reply 1163-4. Epub 2010 Jan 22.

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http://dx.doi.org/10.1016/j.molimm.2009.12.018DOI Listing
May 2010