Molecular targets and signaling pathways regulated by nuclear translocation of syndecan-1.

BACKGROUND: The cell-surface heparan sulfate proteoglycan syndecan-1 is important for tumor cell proliferation, migration, and cell cycle regulation in a broad spectrum of malignancies. Syndecan-1, however, also translocates to the cell nucleus, where it might regulate various molecular functions. RESULTS: We used a fibrosarcoma model to dissect the functions of syndecan-1 related to the nucleus and separate them from functions related to the cell-surface. Nuclear translocation of syndecan-1 hampered the proliferation of fibrosarcoma cells compared to the mutant lacking nuclear localization signal. The growth inhibitory effect of nuclear syndecan-1 was accompanied by significant accumulation of cells in the G0/G1 phase, which indicated a possible G1/S phase arrest. We implemented multiple, unsupervised global transcriptome and proteome profiling approaches and combined them with functional assays to disclose the molecular mechanisms that governed nuclear translocation and its related functions. We identified genes and pathways related to the nuclear compartment with network enrichment analysis of the transcriptome and proteome. The TGF-ß pathway was activated by nuclear syndecan-1, and three genes were significantly altered with the deletion of nuclear localization signal: EGR-1 (early growth response 1), NEK11 (never-in-mitosis gene a-related kinase 11), and DOCK8 (dedicator of cytokinesis 8). These candidate genes were coupled to growth and cell-cycle regulation. Nuclear translocation of syndecan-1 influenced the activity of several other transcription factors, including E2F, NFκß, and OCT-1. The transcripts and proteins affected by syndecan-1 showed a striking overlap in their corresponding biological processes. These processes were dominated by protein phosphorylation and post-translation modifications, indicative of alterations in intracellular signaling. In addition, we identified molecules involved in the known functions of syndecan-1, including extracellular matrix organization and transmembrane transport. CONCLUSION: Collectively, abrogation of nuclear translocation of syndecan-1 resulted in a set of changes clustering in distinct patterns, which highlighted the functional importance of nuclear syndecan-1 in hampering cell proliferation and the cell cycle. This study emphasizes the importance of the localization of syndecan-1 when considering its effects on tumor cell fate.

Characterization and drug sensitivity profiling of primary malignant mesothelioma cells from pleural effusions.

BACKGROUND: Patients with malignant mesothelioma have a poor prognosis and only 40% respond to first line treatment; a combination of pemetrexed and cisplatin or carboplatin. We used primary malignant mesothelioma cells and an ex vivo chemosensitivity assay with future purpose to predict best choice of treatment. The clinical outcome of these patients might be predicted by measuring drug sensitivity. METHODS: Pleural effusions containing primary malignant mesothelioma cells were received from the diagnostic routine. We characterized and tested the chemosensitivity of 18 malignant samples and four benign samples from 16 different patients with pleural effusions. Cells were seeded in a 384-well plate for a robotized ex vivo testing of drug sensitivity to 32 different drugs. The primary cells were further characterized by immunocytochemistry to evaluate the proportion of malignant cells and to study the RRM1 and ERCC1 reactivity, two proteins associated with drug resistance. RESULTS: We observed great individual variability in the drug sensitivity. Primary cell isolates were affected by between one and ten drugs, and resistant to the remaining tested drugs. Actinomycin D and daunorubicin were the two drugs effective in most cases. Adjusting efficiency of individual drugs for varying proportion of tumor cells and to the average effect on benign cells correlated with effect of pemetrexed, cisplatin and survival time. General drug sensitivity, proportion of malignant cells and reactivity to RRM1 correlated to each other and to survival time of the patients. CONCLUSIONS: The proportion of malignant cells and RRM1 reactivity in the pleural effusions correlate to drug sensitivity and survival time. The variability in response to the commonly used chemotherapies emphasizes the need for tests that indicate best individual choice of cytotoxic drugs. The efficiency of the obtained results should preferably be corrected for admixture of benign cells and effects of given drugs on benign cells.

The D ~ Sense ex-vivo viability assay application in a patient with stage IV lung adenocarcinoma: a case report.

BACKGROUND: The treatment resistance is a problem for lung cancer. In this study, we used a vitro tissue culturing system to select a new therapy strategy for a patient with tyrosine kinase inhibitors (TKIs) resistance. CASE PRESENTATION: A 42-year-old male Asian patient was diagnosed with advanced lung adenocarcinoma harboring an exon 19 deletion in the epidermal growth factor receptor (EGFR) gene. The patient was treated with Gefitinib, resulting in an almost complete remission for over a year. The patient relapsed after 13 months treatment, and received four cycles of chemotherapy. At 20 months, the patient had developed multiple lung metastases and a solitary cerebellar metastasis. An EGFR T790M mutation was identified in the peripheral blood sample. Subsequent treatment with Osimertinib resulted in a complete response of the intracranial metastasis. By 33 months, the patient had developed a mediastinal tumor mass that responded well to local radiotherapy. By 39 months, an EGFR C797S cis-mutation had been identified and the patient was treated with Brigatinib and Cetuximab. By 44 months, the tumor cells from the pleural effusion had been tested for sensitivity against 30 targeted and cytostatic drugs using the D ~ Sense ex-vivo viability assay. The assay identified 8 drugs with moderate to high sensitivity. Combination therapy of Gemcitabin and Lobaplatin had resulted in disease stabilization. CONCLUSIONS: The case showed that individualized treatment aided by D ~ Sense ex-vivo viability assay can be a viable option for patients with advanced lung adenocarcinoma with pleural effusions.

Drug sensitivity patterns of HHV8 carrying body cavity lymphoma cell lines.

BACKGROUND: Primary effusion lymphoma (PEL) is a rare KSHV/HHV8-associated high-grade non-Hodgkin's lymphoma (NHL) of B-cell origin, characterized by serous effusions in body cavities. Most patients are HIV-infected men with severe immunosuppression and other HHV8-associated diseases such as Kaposi's sarcoma (KS). The prognosis for those infected is poor, with a median survival of less than 6 months in most cohorts. Sustained complete remission is rare. High-dose chemotherapy regimens are used to improve remission rate and survival. The aim of the present study was to compare the drug sensitivity pattern of the available primary effusion (body cavity based) lymphoma-derived cell lines in order to find additional, potentially effective drugs that are not included in current chemotherapy treatment protocols. METHODS: We have analyzed 11 cell lines against 27 frequently used cytostatic drugs in short term (3 days) survival assays using automated high throughput confocal microscopy. RESULTS: All cell lines showed a distinct, individual drug sensitivity pattern. Considering the in vitro used and clinically achieved drug concentration, Vinorelbine, Paclitaxel, Epirubicin and Daunorubicin were the most effective drugs. CONCLUSIONS: We suggest that inclusion of the above drugs into PEL chemotherapy protocols may be justified. The heterogeneity in the drug response pattern however indicated that assay-guided individualized therapy might be required to optimize therapeutic response.

PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein.

The low molecular weight compound, PRIMA-1MET restores the transcriptional transactivation function of certain p53 mutants in tumor cells. We have previously shown that PRIMA-1MET induces nucleolar translocation of p53, PML, CBP and Hsp70. The Epstein-Barr virus encoded, latency associated antigen EBNA-5 (also known as EBNA-LP) is required for the efficient transformation of human B lymphocytes by EBV. EBNA-5 associates with p53-hMDM2-p14ARF complexes. EBNA-5 is a nuclear protein that translocates to the nucleolus upon heat shock or inhibition of proteasomes along with p53, hMDM2, Hsp70, PML and proteasome subunits. Here we show that PRIMA-1MET induces the nucleolar translocation of EBNA-5 in EBV transformed B lymphoblasts and in transfected tumor cells. The PRIMA-1MET induced translocation of EBNA-5 is not dependent on the presence of mutant p53. It also occurs in p53 null cells or in cells that express wild type p53. Both the native and the EGFP or DSRed conjugated EBNA-5 respond to PRIMA-1MET treatment in the same way. Image analysis of DSRed-EBNA-5 expressing cells, using confocal fluorescence time-lapse microscopy showed that the nucleolar translocation requires several hours to complete. FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) measurements on live cells showed that the nucleolar translocation was accompanied by the formation of EBNA-5 aggregates. The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET. Our results suggest that mutant p53 is not the sole target of PRIMA-1MET. We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET.

Hodgkin-lymphoma-derived cells show high sensitivity to dactinomycin and paclitaxel.

Depending on stage and risk factors, up to 30% of patients with advanced Hodgkin lymphoma (HL) progress or relapse. Patients with pleural effusions have a particularly poor prognosis and this stage of HL is regularly resistant to chemotherapy. All currently available HL cell lines are derived from late stage HL patients. In the present study we measured the sensitivity of these HL lines against the 26 most frequently used cytostatic drugs. We used a novel fluorescent short-term survival assay where the cell was incubated with the drugs for 4 days. The precise number of differentially stained live and dead cells was determined using a custom-built automated laser confocal fluorescent microscope. We found that HL cells, independently of their origin, showed very similar sensitivity patterns for several of the drugs. All HL cell lines were highly sensitive to dactinomycin, paclitaxel and etoposide. Our data suggest that the inclusion of dactinomycin and paclitaxel into chemotherapy protocols against late stage Hodgkin lymphoma with pleural effusion may be justified.

Ex vivo evaluation of tumor cell specific drug responses in malignant pleural effusions.

The effect of chemotherapy may be improved by combining the most effective drugs based on testing the sensitivity of the individual tumor ex vivo. Such estimations of tumor cells from effusions have so far not been implemented in the clinical routine as a basis for individualized choice of therapy. One obstacle for such analyses is the admixture of benign cells that might obscure the results. In this paper we test and compare two ways of performing the analysis specifically on tumor cells. First we enrich the tumor cells, using antibody labeled magnetic separation, and measure the effects of subsequent drug exposure with the metabolic activity assays WST-1 and alamar blue. The second way of estimating drug effects specifically on tumor cells employs multi parameter flow cytometry, measuring apoptosis with the propidium iodide / AnnexinV technique and, particularly for pemetrexed, possible effects on cell cycle progression in immunologically identified tumor cells. The two techniques produce similar results, indicating a possible use in personalized medicine. The possible predictive role of the analysis remains to be shown.

Syndecan-1 in Cancer: Implications for Cell Signaling, Differentiation, and Prognostication.

Syndecan-1, a cell surface heparan sulfate proteoglycan, is critically involved in the differentiation and prognosis of various tumors. In this review, we highlight the synthesis, cellular interactions, and the signalling pathways regulated by syndecan-1. The basal syndecan-1 level is also crucial for understanding the sequential changes involving malignant transformation, tumor progression, and advanced or disseminated cancer stages. Moreover, we focus on the cellular localization of this proteoglycan as cell membrane anchored and/or shed, soluble syndecan-1 with stromal or nuclear accumulation and how this may carry different, highly tissue specific prognostic information for individual tumor types.

Drug sensitivity profiling and molecular characteristics of cells from pleural effusions of patients with lung adenocarcinoma.

We propose to assess the therapeutic value of biomarker-guided individualized chemotherapy in patients with metastasizing lung adenocarcinoma. In this study, we used primary cells from pleural effusions from sixteen patients diagnosed with adenocarcinomas originating in the lung and from four patients with no malignant diagnosis. The ex vivo drug sensitivity of primary cells was assessed for 32 chemotherapeutical drugs. Linear regression analyses were performed to examine possible correlations between the drug sensitivity, overall survival and expression of ERCC1 and RRM1. The ex vivo drug sensitivity profiles of the patients revealed considerable heterogeneity in drug response. Vinblastine, vinorelbine, paclitaxel and actinomycin D showed high efficiency against 50% of the tested primary cells. Significant correlation was detected between the ex vivo sensitivity to platinum based drugs and gemcitabine and the level of ERCC1 and RRM1. No significant correlation was however seen between overall survival and drug sensitivity. The heterogeneity of the drug response suggests that optimal care of the adenocarcinoma patients should include the determination of drug sensitivity of the primary cells and would benefit to use personalized therapy.

Molecular typing of human herpesvirus 8 isolates from patients with Kaposi's sarcoma in Hungary.

BACKGROUND: Kaposi's sarcoma (KS) shows a distinct geographical and ethnic distribution. Genes at both ends of the human herpesvirus 8 (HHV-8) genome have been shown to vary considerably. Seven major molecular subtypes of HHV-8 were defined based on the amino acid sequence of the open reading frame K1 (orf-K1). The aim of the present study was to characterize HHV8 isolates from hospitalized patients in Hungary. MATERIALS AND METHODS: A total of 36 archival paraffin-embedded Kaposi's sarcoma tissue samples were collected. Polymerase chain reaction (PCR) was carried out on the extracted DNA, using specific primers for HHV-8. After identifying the presence of HHV-8 by amplification of its orf26 region, the orf-K1 region was amplified, sequenced and used for phylogenetic analysis. RESULTS: From the 36 orf26-positive cases, orf-K1 was amplified and was analyzed successfully in 12 cases. Phylogenetic studies, based on the complete K1 gene/protein sequences, indicate that all strains belong to the A subtype. Specifically, six of them were related to the A1 subgroup, six to the A2 subgroup and three previously reported to the A3 subgroup. Nucleotide sequence data are reported and are available in the Genbank database under accession numbers KF829938-KF829947.

Chronic lymphoid leukemia cells are highly sensitive to the combination of prednisolone and daunorubicin, but much less to doxorubicin or epirubicin.

OBJECTIVE: To generate a comprehensive map of the drug sensitivity of chronic lymphoid leukemia cells (CLL) using a newly developed in vitro drug-sensitivity assay based on automated evaluation of cell viability on single-cell level. MATERIALS AND METHODS: Primary CLL cells from 77 patients were tested using automated digital fluorescence microscopy. The effect of 27 frequently used chemotherapeutic agents was measured in short-term fluorescence survival assay. To avoid typical in vitro artifacts such as growth factor depletion and oxidative damage, the cell were cultured in a novel, total human blood lysate-based medium (OmniSanguine) in order to preserve the composition of growth factor flora and redox conditions of the in vivo environment. RESULTS: CLL cells from different patients showed considerable heterogeneity in their drug-sensitivity patterns. This pattern was stable even after in vitro activation of cell proliferation. Half of the samples were sensitive to fludarabine and chlorambucil. Daunorubicin was the most potent drug. It was effective in 75 of 77 cases. In addition, daunorubicin and prednisolone showed a strong synergistic effect. CONCLUSIONS: We suggest that the combination of low-dose daunorubicin and prednisolone might be an additional treatment option for therapy-resistant cases of CLL.

NK cell-mediated lysis is essential to kill Epstein-Barr virus transformed lymphoblastoid B cells when using rituximab.

Rituximab is a humanized chimeric monoclonal antibody, targeted against the pan B cell marker CD20. It is frequently used to treat a variety of B cell lymphomas and immunosuppression associated lymphoproliferations such as posttransplant lymphoproliferative disorder (PTLD). The response rate of rituximab treatment is 65%, but the exact in vivo mechanism of action is not yet fully understood, although antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct induction of apoptosis have been suggested as effector mechanism. Rituximab may affect different types of lymphomas through different mechanisms. As lymphoblastoid cell lines (LCLs) are well-established in vitro models of PTLD, we investigated the effect of rituximab on these cells using a custom built automated laser confocal fluorescent microscope. We found that rituximab alone was not effective at inducing cell death of EBV-transformed B cells. The antibody was effective in the complement-mediated CDC. Rituximab could induce NK cell-mediated ADCC but it was more effective in the presence of untreated fresh human plasma compared to heat-inactivated human plasma. Our data suggest that complement-enhanced NK-mediated ADCC is required for effective rituximab mediated killing of EBV-transformed B cells. Determining and monitoring of serum complement levels and in vitro killing efficacy of NK cells of PTLD patients might help to predict resistant cases to rituximab therapy. On the other hand our results suggest a possibility that rituximab should be combined only with cytotoxic drugs that spare NK function when treating PTLD patients.

Effect of frequently used chemotherapeutic drugs on cytotoxic activity of human cytotoxic T-lymphocytes.

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded cytotoxic T-lymphocytes (CTL). It is important to consider the drug-induced effects when chemotherapeutic regimens and CTL-mediated immunotherapy is planned to be used in parallel. In this study, we characterized the effect of 29 frequently used chemotherapeutic agents on the cytotoxic activity of autologous and allogeneic CTLs. We found that treatment of CTLs with the following drugs: docetaxel, vincristine, chlorambucil, mitomycin C, oxaliplatin, doxorubicin, and bleomycin effectively inhibited CTL-mediated killing, without affecting their viability. On the other hand, the following drugs enhanced or permitted efficient CTL-mediated killing in vitro at concentrations comparable with the maximally achieved therapeutic concentration in vivo in humans: daunorubicin, prednisolone, vinorelbine, cisplatin, methotrexate, hydroxyurea, cytarabine, cyclophosphamide, topotecan, epirubicin, fluorouracil, carboplatin, asparaginase, 6-mercaptopurine, and bortezomib. Our results could potentially be used in the future to design new CTL-based adjuvant immunotherapy protocols.