Phosphorylated STAT3 physically interacts with NPM and transcriptionally enhances its expression in cancer.

The signal transducer and activator of transcription 3 (STAT3) can be activated by the tyrosine kinase domain of the chimeric protein nucleophosmin/anaplastic lymphoma kinase (NPM/ALK), and has a pivotal role in mediating NPM/ALK-related malignant cell transformation. Although the role of STAT3 and wild-type NPM in oncogenesis has been extensively investigated, the relationship between both molecules in cancer remains poorly understood. In the present study, we first demonstrate that STAT3 phosphorylation at tyrosine 705 is accompanied by a concomitant increase in the expression level of NPM. Nuclear co-translocation of phosphorylated STAT3 with NPM can be triggered by interferon-alpha (IFN-α) stimulation of Jurkat cells and phosphorylated STAT3 co-localizes with NPM in cancer cells showing constitutive STAT3 activation. We further demonstrate that STAT3 phosphorylation can transcriptionally mediate NPM upregulation in IFN-α-stimulated Jurkat cells and is responsible for maintaining its expression in cancer cells showing constitutive STAT3 activation. Inhibition of STAT3 phosphorylation or knockdown of NPM expression abrogates their simultaneous transnuclear movements. Finally, we found evidence for a physical interaction between NPM and STAT3 in conditions of STAT3 activation. In conclusion, NPM is a downstream effector of the STAT3 signaling, and can facilitate the nuclear entry of phosphorylated STAT3. These observations might open novel opportunities for targeting the STAT3 pathway in cancer.

Sequence evolution and escape from specific immune pressure of an HIV-1 Rev epitope with extensive sequence similarity to human nucleolar protein 6.

Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8(+) T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8(+) T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4(+) T-cell count and viral load remained stable despite escape from T-cell recognition.

Fighting with the enemy's weapons? the role of costimulatory molecules in HIV.

HIV infection is characterized by a number of abnormalities in several components of the immune system. For example, during HIV infection, a massive decrease of CD4(+) T cells is observed, as well as a progressive depletion of naïve CD8(+) T cells. Furthermore, elevated numbers of apoptotic B and T cells are present in HIV-infected patients, and a systemic immune activation results in T-cell exhaustion. Finally, HIV infection is characterized by the presence of functionally impaired dendritic cells, with decreased expression of maturation markers, decreased secretion of cytokines and defects in antigen processing and presentation. All these characteristics result in the occurrence of non-functional cytotoxic T lymphocytes, that fail to control HIV-replication in most individuals during progressive disease. Costimulatory and co-inhibitory molecules are involved in the activation, differentiation and survival of several cell-types of the immune system. Each costimulatory receptor (generally expressed on effector cells) can conjugate with one or more specific ligands (expressed on antigen-presenting cells), which leads to an activation of intracellular signaling pathways inside the cells on which they are expressed. HIV infection is characterized by an aberrant expression of these molecules on cells of the immune system. Many of the immune deficiencies mentioned in the previous paragraph can be explained by abnormal expression of costimulatory molecules, and could consequently be overcome by interfering with their interactions. In this review, we give an overview of the functions and expression patterns of the receptor/ligand pairs of the tumor necrosis factor and the B7 super-families of costimulatory and co-inhibitory molecules in HIV-infected patients. We will also discuss possibilities for manipulating their signaling as a therapeutic anti-HIV tool.

Oncolytic adenoviral mutants induce a novel mode of programmed cell death in ovarian cancer.

Oncolytic adenoviral mutants have considerable activity in ovarian cancer. However, the mechanisms by which they induce cell death remain uncertain. dl922-947, which contains a 24 bp deletion in E1A CR2, is more potent than both E1A wild-type adenoviruses and the E1B-55K deletion mutant dl1520 (Onyx-015). We investigated the mode of death induced by three E1A CR2-deleted replicating adenoviruses in models of ovarian cancer and also the importance of E3 11.6 (adenovirus death protein) in determining this mode of death. Ovarian cancer cells were infected with dl922-947 (E3 11.6+) and dlCR2 (E3 11.6-). We also generated dlCR2 tSmac, which also encodes the gene for processed Smac/DIABLO. Classical apoptosis does not occur in adenoviral cell death and there is no role for mitochondria. Expression of Smac/DIABLO does not enhance cytotoxicity nor increase apoptotic features. A role for cathepsins and lysosomal membrane permeability was excluded. Autophagy is induced, but is not the mode of death and may act as a cell survival mechanism. There is no evidence of pure necrosis, while the presence of E3 11.6 does not modulate the mode or extent of cell death. Thus, E1A CR2-deleted oncolytic adenoviral cytotoxicity in ovarian cancer may define a novel mode of programmed cell death.

Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics.

Lentiviral vectors have emerged as promising tools for both gene therapy and immunotherapy purposes. They exhibit several advantages over other viral systems in that they are less immunogenic and are capable of transducing a wide range of different cell types, including dendritic cells (DC). DC transduced ex vivo with a whole range of different (tumor) antigens were capable of inducing strong antigen-specific T-cell responses, both in vitro and in vivo. Recently, the administration of lentiviral vectors in vivo has gained substantial interest as an alternative method for antigen-specific immunization. This method offers a number of advantages over DC vaccines as the same lentivirus can in principle be used for all patients resulting in a significantly reduced cost and requirement for considerably less expertise for the generation and administration of lentiviral vaccines. By selectively targeting lentiviral vectors to, or restricting transgene expression in certain cell types, selectivity, safety and efficacy can be further improved. This review will focus on the use of direct administration of lentiviral vectors encoding tumor-associated antigens (TAA) for the induction of tumor-specific immune responses in vivo, with a special focus on problems related to the generation of large amounts of highly purified virus and specific targeting of antigen-presenting cells (APC).

Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA.

The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response. Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation. Co-electroporation of DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail. These results indicate that DC co-electroporation with both dsRNA and tumor antigen encoding mRNA induces fully activated and antigen-loaded DCs that promote antigen-specific CTL responses and may provide the basis for future immunotherapeutic strategies.

Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors.

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.

Cancer cell dissemination during curative surgery for colorectal liver metastases.

AIM: The amount of cancer cells disseminated during curative surgery for colorectal liver metastases (CRLM) may be responsible for recurrence. Haematogenous and intrahepatic cancer cell dissemination was evaluated, and its impact on cancer recurrence was assessed. METHOD: Twenty patients with resectable CRLM were included in a prospective study. Twelve patients underwent curative resection for 21 metastases. Ten selected metastases in eight patients were treated with radiofrequency ablation (RFA) followed by resection at the same operative session. Cancer cell dissemination was determined before, during and after surgery using 'real time' quantitative RT-PCR assay, based on detection and quantification of CEA and CK20 mRNA transcripts. RESULTS: Circulating cancer cells were detected in 80% and intrahepatic cancer cells in 37% of the patients, though without impact on cancer recurrence. The amounts of disseminated cancer cells were significantly increased after surgery. This increase was similar in patients treated with and without RFA. RFA caused complete tumour destruction. CONCLUSION: Curative surgery for CRLM significantly increases the amount of disseminated cancer cells. Radiofrequency ablation can completely destroy selected resectable CRLM without excessive cancer cell dissemination. Neither haematogenous nor intrahepatic cancer cell dissemination were related to cancer recurrence in this small patient series.

Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytic leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n=278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

Real-time quantitative RT-PCR and detection of tumour cell dissemination in breast cancer patients: plasmid versus cell line dilutions.

BACKGROUND: We previously developed a real-time quantitative RT-PCR technique to detect breast carcinoma cells in peripheral blood (PB). The aim of the current study was to improve cytokeratin 19 (CK19) quantification using plasmid dilutions of cloned PCR fragments to obtain a more reliable and reproducible quantification of CK19 transcripts. MATERIALS AND METHODS: PB samples of 14 stage IV breast cancer patients and 23 healthy controls were examined with RT-PCR using plasmid quantification. RESULTS: Median CK19+ copy numbers of one and 11 were detected in the control group and stage IV breast cancer patients, respectively (Mann-Whitney, P