CD59

Gene Summary

Gene:CD59; CD59 molecule
Aliases: 1F5, EJ16, EJ30, EL32, G344, MIN1, MIN2, MIN3, MIRL, HRF20, MACIF, MEM43, MIC11, MSK21, 16.3A5, HRF-20, MAC-IP, p18-20
Location:11p13
Summary:This gene encodes a cell surface glycoprotein that regulates complement-mediated cell lysis, and it is involved in lymphocyte signal transduction. This protein is a potent inhibitor of the complement membrane attack complex, whereby it binds complement C8 and/or C9 during the assembly of this complex, thereby inhibiting the incorporation of multiple copies of C9 into the complex, which is necessary for osmolytic pore formation. This protein also plays a role in signal transduction pathways in the activation of T cells. Mutations in this gene cause CD59 deficiency, a disease resulting in hemolytic anemia and thrombosis, and which causes cerebral infarction. Multiple alternatively spliced transcript variants, which encode the same protein, have been identified for this gene. [provided by RefSeq, Jul 2008]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:CD59 glycoprotein
Source:NCBIAccessed: 09 March, 2017

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 10 March 2017 using data from PubMed using criteria.

Literature Analysis

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Tag cloud generated 09 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CD59 (cancer-related)

Horibata K, Ukai A, Ishikawa S, et al.
Monitoring genotoxicity in patients receiving chemotherapy for cancer: application of the PIG-A assay.
Mutat Res Genet Toxicol Environ Mutagen. 2016; 808:20-6 [PubMed] Related Publications
The recently introduced Pig-a in vivo gene mutation assay measures endogeneous mutations of Pig-a (human, PIG-A), an X-linked gene that is conserved across species from rodents to humans. Flow cytometric analysis enables the enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient erythrocytes, resulting from a mutation in Pig-a/PIG-A, in only a few microliters of peripheral blood. Pig-a/PIG-A mutations appear to function in a neutral manner, allowing evaluation of the accumulated genotoxic effects of repeated exposures. To date, most Pig-a studies have been conducted in rodents; only a few reports regarding human applications of the PIG-A assay have been published. We have conducted a PIG-A assay in the context of human genotoxicity monitoring. Peripheral blood was collected from healthy human donors and chemotherapy-treated cancer patients at Yamagata University Hospital. To investigate the PIG-A mutant frequency (MF) induced by chemotherapy, red blood cells were analyzed via flow cytometry following staining with allophycocyanin-conjugated anti-CD235ab (erythrocyte specific) and fluorescein isothiocyanate-conjugated anti-CD59 antibodies (GPI-anchored protein specific). Reticulocyte frequencies (%RET) were also analyzed using a phycoerythrin-conjugated anti-CD71 antibody to monitor bone marrow suppression and reticulocytosis. Two of 27 patients exhibited a significantly elevated frequency of PIG-A mutants. Although we observed either a reduced or an increased %RET in all patients, no association was observed between this factor and the PIG-A MF. Unfortunately, we could not analyze blood samples collected before treatment during therapeutic processes. Additionally, the sampling time point for some patients was too short to express the PIG-A mutant phenotypes. Therefore, the possibility of natively high PIG-A MFs prior to treatment must be considered. The human PIG-A assay shows promise as a human genotoxicity monitoring method.

Ouyang Q, Zhang L, Jiang Y, et al.
The membrane complement regulatory protein CD59 promotes tumor growth and predicts poor prognosis in breast cancer.
Int J Oncol. 2016; 48(5):2015-24 [PubMed] Related Publications
Breast cancer is the most prevalent type of cancer among women. CD59, a membrane complement regulatory protein, has been demonstrated to be overexpressed in most solid tumors, where it facilitates tumor cell escape from complement surveillance. However, the role of CD59 in breast cancer growth and clinical prognosis is not fully revealed. To investigate the role of CD59 in breast cancer growth and prognostic significance, we knocked down CD59 in a breast cancer cell line that is highly metastatic to the lungs, MDA-MB‑231-HM. Cell growth was measured in vitro and in vivo using a xenograft model. In addition, clinical data on a cohort of 120 patients with or without lung metastasis was analyzed based on CD59 expression, which was detected by immunohistochemistry. Knockdown of CD59 significantly inhibited MDA-MB‑231-HM cell growth both in vitro and in vivo. An analysis of clinical data on 120 patients revealed that patients with CD59 overexpression may have a worse prognosis. CD59 may therefore be a prognostic biomarker for poor outcome in breast cancer patients.

Chen X, Xu Y, Cao X, et al.
Associations of Il-1 Family-Related Polymorphisms With Gastric Cancer Risk and the Role of Mir-197 In Il-1f5 Expression.
Medicine (Baltimore). 2015; 94(47):e1982 [PubMed] Free Access to Full Article Related Publications
To explore whether the roles of IL-1 family single nucleotide polymorphisms (SNPs) of the microRNA binding sites (miR-SNPs) in the 3' untranslated region (3'-UTR) of their target genes in the progression of gastric cancer (GC) and verify the relationship between miR-197 with chronic inflammatory gene-IL1-F5 by microRNA target prediction, a case-control study which consisted of 500 cases and 500 frequency-matched healthy controls was conducted. Single nucleotide polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or allele-specific PCR (AS-PCR). Association between SNPs and GC risk was evaluated by adjusted odds ratios (ORs) and 95% confidence intervals (CIs) in unconditional logistic regression analyses. Quantitative real-time (qRT) PCR assay and Western Blot analyses were performed to analyze the miR-197 expression and the IL1-F5 expression. The variant homozygote and heterozygote genotype of rs9005 in IL-1RN were significantly associated with increased risks of GC (ORadjusted [95%CI]: 1.71[1.04-2.81] and ORadjusted[95%CI]: 1.36 [1.04-1.78]). Compared with the wild heterozygote genotype, the variant heterozygote genotype of rs2472188 and rs2515401 in IL-1F5 polymorphisms were significantly associated with increased GC risks (ORadjusted [95%CI]: 1.51[1.15-1.99] and ORadjusted[95%CI]: 1.36[1.04-1.76]), but no significant differences existed in other 7 IL-1 family SNPs (rs2856836 in IL-1A, rs3732131 in IL-1R1, rs1135354 and rs3771157 in IL-18RA, rs3180235, rs957201 and rs2515402 in IL-1F5) with GC. The recombinant plasmid-pGenesil-1-miR-197 could upregulate the expression of miR-197 and downregulate the expression of IL-1F5 in human gastric cancer cell lines SGC-7901 and BGC-823 cells after transfection, and the miR-197 inhibitor could facilitate the expression of IL1-F5 after transfecting the same cell lines. These results suggested that SNPs in the IL-1 family genes play important roles in the development of GC and the IL-1F5 might be the target gene of miR-197, and miR-197 might negatively regulate its expression.

Kapka-Skrzypczak L, Wolinska E, Szparecki G, et al.
CD55, CD59, factor H and factor H-like 1 gene expression analysis in tumors of the ovary and corpus uteri origin.
Immunol Lett. 2015; 167(2):67-71 [PubMed] Related Publications
The expression level of complement regulators in ovarian and corpus uteri tumors was not fully established so far. In current manuscript we performed gene expression analysis by the real-time PCR approach to investigate both membrane bound - CD55 and CD59 and fluid phase - factor H and factor H-like 1 complement regulators. We found increased CD55 expression in corpus uteri tumors when compared to control tissues, whereas in ovarian cancer CD55 expression was lower than in control sections. Additionally we found CD59 expression to be more prominent in ovarian cancer than in corpus uteri tumor samples. We observed also the strong positive correlation between the level of expression of the whole group of regulators, which was particularly significant between the expression of factor H and factor H- like 1. In conclusion we present novel results which implicates different role of particular complement inhibitors in the regulation of the complement system in two cancer types examined. Strong positive correlation between examined proteins implicates similar pattern of the regulation which should be taken into consideration with regards to the possible immunotherapy applied as adjuvant therapeutic approach in these two indications. The inhibition of complement regulation may serve as a strategy to potentiate the efficacy of such treatment.

Chatziantoniou V, Alexia S, Konstantopoulos K, et al.
Significance of the detection of paroxysmal nocturnal hemoglobinuria clones in patients with multiple myeloma undergoing autologous stem cell transplantation.
Hematol Oncol Stem Cell Ther. 2015; 8(4):150-9 [PubMed] Related Publications
OBJECTIVE/BACKGROUND: There are reports about the presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in multiple myeloma (MM), but these have been demonstrated only in red blood cells (RBCs) and the previous reports utilized an obsolete diagnostic method. We carried out a study to identify the clones by flow cytometry (FC) and to understand their clinical significance.
METHODS: A prospective study on consecutive patients with newly diagnosed MM who were candidates for autologous stem cell transplantation (ASCT) from 2008 to 2012. We screened peripheral blood samples by FC for CD55- and/or CD59-deficient RBC, neutrophils, and monocytes. PNH testing was carried out at diagnosis, before ASCT and 3 months after ASCT, as well as sporadically during MM remission and at disease relapse.
RESULTS: A total of 31 patients were included in the study. PNH clones reaching a median size of 10.8% (range 4.0-18.7%) were found in 10 patients (32.3%). Clones were detected at diagnosis in nine patients and 3 months after ASCT in one patient. A correlation between the presence of the clones and subclinical hemolysis was observed. Nevertheless, the presence of the clones did not influence the overall management and prognosis of the patients.
CONCLUSION: We confirmed findings of previous reports with current diagnostic guidelines and showed that although the size of the clones may be relatively large, their presence is probably not detrimental. The clinical significance of these clones and the possible mechanisms underlying their expansion in MM must be a subject of further investigation.

Goswami MT, Reka AK, Kurapati H, et al.
Regulation of complement-dependent cytotoxicity by TGF-β-induced epithelial-mesenchymal transition.
Oncogene. 2016; 35(15):1888-98 [PubMed] Free Access to Full Article Related Publications
The process of epithelial-mesenchymal transition (EMT), in addition to being an initiating event for tumor metastasis, is implicated in conferring several clinically relevant properties to disseminating cancer cells. These include stem cell-like properties, resistance to targeted therapies and ability to evade immune surveillance. Enrichment analysis of gene expression changes during transforming growth factor-β (TGF-β)-induced EMT in lung cancer cells identified complement cascade as one of the significantly enriched pathway. Further analysis of the genes in the complement pathway revealed an increase in the expression of complement inhibitors and a decrease in the expression of proteins essential for complement activity. In this study, we tested whether EMT confers resistance to complement-dependent cytotoxicity (CDC) in lung cancer cells and promotes tumor progression. CD59 is a potent inhibitor of membrane attack complex that mediates complement-dependent cell lysis. We observed a significant increase in the CD59 expression on the surface of cells after TGF-β-induced EMT. Furthermore, CD59 knockdown restored susceptibility of cells undergoing EMT to cetuximab-mediated CDC. TGF-β-induced CD59 expression during EMT is dependent on Smad3 but not on Smad2. Chromatin immunoprecipitation analysis confirmed that Smad3 directly binds to the CD59 promoter. Stable knockdown of CD59 in A549 cells inhibited experimental metastasis. These results demonstrate that TGF-β-induced EMT and CD59 expression confers an immune-evasive mechanism to disseminating tumor cells facilitating tumor progression. Together, our data demonstrates that CD59 inhibition may serve as an adjuvant to enhance the efficacy of antibody-mediated therapies, as well as to inhibit metastasis in lung cancer.

Marquez ME, Hernández-Uzcátegui O, Cornejo A, et al.
Bone marrow stromal mesenchymal cells induce down regulation of CD20 expression on B-CLL: implications for rituximab resistance in CLL.
Br J Haematol. 2015; 169(2):211-8 [PubMed] Related Publications
Although the majority of B cells express surface CD20 in chronic lymphocytic leukaemia (B-CLL), only ∼50% of patients respond to treatment with rituximab. Decreased CD20 expression on these tumour B cells could be responsible for the lack of response observed in some patients treated with rituximab. Despite the potential critical role of CD20 in the biology of B cell malignancies, the mechanisms controlling its expression are poorly understood. At the bone marrow level, mesenchymal stromal cells (MSC) may regulate and support the survival of malignant cells, such as B-CLL cells. In this study, we investigated whether MSC may regulate the CD20 expression on B-CLL. For this purpose, B cells from CLL patients were isolated and co-cultured on MSC. B-CLL cells were collected from B-CLL/MSC co-cultures and examined for their expression of CD20. We demonstrate decreased CD20 expression in B-CLL cells after 2 weeks of co-culture with MSC, under contact and non-contact conditions, which was associated with a decreased susceptibility to rituximab. Additionally, B cells co-cultured with MSCs show an increase in CD59 expression. Our findings strongly suggest that the interaction between B-CLL cells and MSC may play a major role in the resistance to rituximab-induced apoptosis of B-CLL cells.

Liu M, Yang YJ, Zheng H, et al.
Membrane-bound complement regulatory proteins are prognostic factors of operable breast cancer treated with adjuvant trastuzumab: a retrospective study.
Oncol Rep. 2014; 32(6):2619-27 [PubMed] Related Publications
Complement-dependent cytotoxicity (CDC) is an important antitumor mechanism of monoclonal antibodies (mAbs). However, trastuzumab, an anti-HER2 mAb, exerts only minor CDC. Overexpression of membrane-bound complement regulatory proteins (mCRPs), which suppress CDC, have been implicated in various malignant tumors. Here, we explored the predictive role of the expression levels of three mCRPs (CD55, CD59 and CD46) in the prognosis of breast cancer cases that underwent adjuvant trastuzumab treatment. We also studied the effect of mCRP downregulation on trastuzumab-induced CDC in vitro. Sixty-five HER2-positive breast cancer patients who received adjuvant therapy containing trastuzumab, were retrospectively analyzed. Levels of CD55, CD59 and CD46 expression were detected by immunohistochemistry. Chi-square test, Kaplan‑Meier survival analysis and a Cox proportional hazards model were used to analyze the association between CD55, CD59 and CD46 expression and prognosis. HER2-positive SK-Br3 and BT-474 breast cancer cells were pretreated with various drugs to reduce mCRP expression. Afterwards, trastuzumab‑mediated cytolytic effects were measured. Among the 65 patients, 46.2% had high expression of CD55, 44.6% had high expression of CD59 and 44.6% had high expression of CD46. The median follow-up duration was 47 months (range from 24 to 75 months). Patients with CD55 or CD59 overexpression had a higher relapse rate than those with low expression of CD55 (33.3 vs. 8.6%; P=0.013) or CD59 (31.0 vs. 11.1%; P=0.046). Similarly, mean disease-free survival of patients with CD55 or CD59 overexpression was significantly shorter than those with a low expression of CD55 (56 vs. 70 months; log-rank test, P=0.008) or CD59 (56 vs. 69 months; log-rank test, P=0.033). Multivariate analysis confirmed that CD55, but not CD59, was an independent risk factor of recurrence (HR=4.757; 95% CI, 0.985-22.974; P=0.05). In vitro, we found that tamoxifen inhibited both the protein and mRNA expression levels of CD55, but not CD59 or CD46 in SK-Br3 and BT-474 cells. After pretreatment of tamoxifen, trastuzumab-induced cytolysis was enhanced through CD55 downregulation. In conclusion, CD55 overexpression is an independent risk factor for recurrence in breast cancer patients receiving postoperative adjuvant therapy containing trastuzumab. Combined use of tamoxifen and trastuzumab for HER2-positive breast cancer treatment may enhance the antitumor effects of trastuzumab by elevated CDC, which warrants further study.

Song G, Song G, Ni H, et al.
Deregulated expression of miR-224 and its target gene: CD59 predicts outcome of diffuse large B-cell lymphoma patients treated with R-CHOP.
Curr Cancer Drug Targets. 2014; 14(7):659-70 [PubMed] Related Publications
miRNAs are non-coding RNA molecules; their deregulations may contribute to cancer pathogenesis. However, the mechanisms of how miRNA dysfunction contributes to the lymphomagenesis of diffuse large B-cell lymphoma (DLBCL) are not well established. In this study, we analyzed the expression of miR-224 in four DLBCL cell lines and 168 patients' specimens. We found that the expression of miR-224 in DLBCL was down-regulated compared with normal B-cell but was not statistically different between the germinal center B-cell-like-type and the activated B-cell-like-type. Using bioinformatics prediction and luciferase report assays, we demonstrated that miR-224 directly down-regulated CD59 expression by binding to its 3'-untranslated region. We also used immunohistochemical staining of CD59 in human DLBCL specimens and analyzed the relationship between the expression of miR-224, CD59 and the overall/progress-free survival of DLBCL patients who were uniformly treated with rituximab,cyclophosphamide, adriamycin, vincristine, and prednisone (R-CHOP). We found that miR-224 may contribute to DLBCL pathogenesis. Most importantly, the expression of miR-224 and CD59 can predict the response and outcome of DLBCL patients treated with R-CHOP.

Rösner T, Lohse S, Peipp M, et al.
Epidermal growth factor receptor targeting IgG3 triggers complement-mediated lysis of decay-accelerating factor expressing tumor cells through the alternative pathway amplification loop.
J Immunol. 2014; 193(3):1485-95 [PubMed] Related Publications
Binding of C1q to target-bound IgG initiates complement-mediated lysis (CML) of pathogens, as well as of malignant or apoptotic cells, and thus constitutes an integral part of the innate immune system. Despite its prominent molecular flexibility and higher C1q binding affinity compared with human IgG1, IgG3 does not consistently promote superior CML. Hence the aim of this study was to investigate underlying molecular mechanisms of IgG1- and IgG3-driven complement activation using isotype variants of the therapeutic epidermal growth factor receptor (EGFR) Ab cetuximab. Both IgG1 and IgG3 Abs demonstrated similar EGFR binding and similar efficiency in Fab-mediated effector mechanisms. Whereas anti-EGFR-IgG1 did not promote CML of investigated target cells, anti-EGFR-IgG3 triggered significant CML of some, but not all tested cell lines. CML triggered by anti-EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement regulatory proteins CD55 and CD59, but not CD46. Notably, anti-EGFR-IgG3 promoted strong C1q and C3b, but relatively low C4b and C5b-9 deposition on analyzed cell lines. Furthermore, anti-EGFR-IgG3 triggered C4a release on all cells but failed to induce C3a and C5a release on CD55/CD59 highly expressing cells. RNA interference-induced knockdown or overexpression of membrane-bound complement regulatory proteins revealed CD55 expression to be a pivotal determinant of anti-EGFR-IgG3-triggered CML and to force a switch from classical complement pathway activation to C1q-dependent alternative pathway amplification. Together, these data suggest human anti-EGFR-IgG3, although highly reactive with C1q, to weakly promote assembly of the classical C3 convertase that is further suppressed in the presence of CD55, forcing human IgG3 to act mainly through the alternative pathway.

Sebejova L, Borsky M, Jaskova Z, et al.
Distinct in vitro sensitivity of p53-mutated and ATM-mutated chronic lymphocytic leukemia cells to ofatumumab and rituximab.
Exp Hematol. 2014; 42(10):867-74.e1 [PubMed] Related Publications
Abnormalities in ATM and TP53 genes represent important predictive factors in chronic lymphocytic leukemia (CLL); however, the efficacy of CD20 targeting immunotherapy is only poorly defined in the affected patients. Therefore, we tested the in vitro response to ofatumumab (OFA) and rituximab (RTX) in 75 CLL samples with clearly defined p53 or ATM inactivation. Using standard conditions allowing complement-dependent cytotoxicity, i.e., 10 μg/mL of antibodies and 20% active human serum, we observed clear differences among the tested genetic categories: ATM-mutated samples (n = 17) represented the most sensitive, wild-type samples (n = 31) intermediate, and TP53-mutated samples (n = 27) the most resistant group (ATM-mut vs. TP53-mut: P = 0.0005 for OFA and P = 0.01 for RTX). The response correlated with distinct levels of CD20 and critical complement inhibitors CD55 and CD59; CD20 level median was the highest in ATM-mutated and the lowest in TP53-mutated samples (difference between the groups P < 0.01), while the total level of complement inhibitors (CD55 plus CD59) was distributed in the opposite manner (P < 0.01). Negligible response to both OFA and RTX was noted in all cultures (n = 10) tested in the absence of active serum, which strongly indicated that complement-dependent cytotoxicity was a principal cell death mechanism. Our study shows that (1) common genetic defects in CLL cells significantly impact a primary response to anti-CD20 monoclonal antibodies and (2) ATM-mutated patients with currently poor prognosis may potentially benefit from immunotherapy targeting CD20.

Kesselring R, Thiel A, Pries R, et al.
The complement receptors CD46, CD55 and CD59 are regulated by the tumour microenvironment of head and neck cancer to facilitate escape of complement attack.
Eur J Cancer. 2014; 50(12):2152-61 [PubMed] Related Publications
BACKGROUND: Membrane-bound complement restriction proteins (mCRPs) CD46, CD55 and CD59 enable tumour cells to evade complement dependent cytotoxicity and antibody-dependent killing mechanisms. But less is known about the role of these mCRPs in head and neck cancer.
METHODS: In this study we determined the expression of the mCRPs on head and neck squamous cell carcinoma (HNSCC) cell lines, on tumour tissue and TDLNs (tumour-draining lymph nodes) as well as on lymphocytes from HNSCC patients. The influence of the HNSCC microenvironment on the mCRP regulation was analysed using Flow Cytometry, Western blotting and small interfering RNAs (siRNA) transfection studies.
RESULTS: We examined the effects of the HNSCC tumour milieu on the expression levels of CD46, CD55 and CD59. We investigated the susceptibility of HNSCC cells to CDC (complement-dependent cytotoxicity) while silencing the mCRPs. Our results demonstrate a huge influence of the HNSCC tumour microenvironment on the regulation of mCRP expression and show a reciprocal regulation between the different mCRPs themselves.
CONCLUSIONS: In summary, our data indicate that HNSCC has evolved different strategies to evade complement attacks and that the tumour microenvironment leads to the enhancement of complement resistance of the surrounding tissue.

Dertinger SD, Avlasevich SL, Torous DK, et al.
Persistence of cisplatin-induced mutagenicity in hematopoietic stem cells: implications for secondary cancer risk following chemotherapy.
Toxicol Sci. 2014; 140(2):307-14 [PubMed] Free Access to Full Article Related Publications
Cisplatin is a cytostatic agent used in the treatment of many types of cancer, but its use is associated with increased incidences of secondary leukemia. We evaluated cisplatin's in vivo genotoxic potential by analyzing peripheral blood for Pig-a mutant phenotype erythrocytes and for chromosomal damage in the form of micronuclei. Mutant phenotype reticuloyte and erythrocyte frequencies, based on anti-CD59 antibody labeling and flow cytometric analysis, were determined in male Sprague Dawley rats treated for 28 consecutive days (days 1-28) with up to 0.4 mg cisplatin/kg/day, and sampled on days -4, 15, 29, and 56. Vehicle and highest dose groups were evaluated at additional time points post-treatment up to 6 months. Day 4 and 29 blood samples were also analyzed for micronucleated reticulocyte frequency using flow cytometry and anti-CD71-based labeling. Mutant phenotype reticulocytes were significantly elevated at doses ≥0.1 mg/kg/day, and mutant phenotype erythrocytes were elevated at doses ≥0.05 mg/kg/day. In the 0.4 mg/kg/day group, these effects persisted for the 6 month observation period. Cisplatin also induced a modest but statistically significant increase in micronucleus frequency at the highest dose tested. The prolonged persistence in the production of mutant erythrocytes following cisplatin exposure suggests that this drug mutates hematopoietic stem cells and that this damage may ultimately contribute to the increased incidence of secondary leukemias seen in patients cured of primary malignancies with platinum-based regimens.

Modlin IM, Drozdov I, Kidd M
Gut neuroendocrine tumor blood qPCR fingerprint assay: characteristics and reproducibility.
Clin Chem Lab Med. 2014; 52(3):419-29 [PubMed] Related Publications
BACKGROUND: We have developed a PCR-based tool that measures a 51-gene panel for identification of gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) in peripheral blood. This manuscript assesses the robustness (performance metrics) of this tool with a specific focus on the effects of individual parameters including collection, storage, acid suppressive medication [proton pump inhibitor (PPI)], age, sex, race and food on accuracy.
METHODS: Performance metrics were evaluated using a gold standard (mRNA derived from three individual human neuroendocrine tumor cell lines) and clinical samples using qPCR.
RESULTS: One hundred percent of the 51 transcripts were amplified in the gold standard (NEN cell line-derived mRNA) (CQ<35, average efficiency 1.94). The inter- and intra-assay variations were 1%-2%. In clinical samples, 50 of 51 targets (98%) were amplified. The inter- and intra-assay reproducibility ranged between 0.4% and 1.2%. The coefficient of variation (CV) was 5.3%. Expression of the reference gene, ALG9, was robust [low variation, low M-value, high (99.5%) PCR efficiency] and unaffected by sample processing. Test meals, long-term PPI use (>1 year), age, sex and ethnicity had no effect on the signature. Expression of two genes, ALP2 and CD59 correlated strongly with RNA integrity (R=0.72, p<0.001) and could be used to assess storage and processing.
CONCLUSIONS: The 51 marker gene signature was robust and reproducible, exhibiting acceptable inter- and intra-assay metrics (<5%). Feeding, PPI intake, age, sex and ethnicity do not affect the signature. Expression levels of APLP2 and CD59 are effective surrogate markers of proper sample collection and processing.

Li B, Lin H, Fan J, et al.
CD59 is overexpressed in human lung cancer and regulates apoptosis of human lung cancer cells.
Int J Oncol. 2013; 43(3):850-8 [PubMed] Related Publications
CD59, belonging to membrane complement regulatory proteins (mCRPs), inhibits the cytolytic activity of complement and is overexpressed in many types of solid cancers. The aim of the present study was to detect the expression of CD59 in non-small cell lung cancer (NSCLC) and to investigate the relationship between decreased CD59 expression and tumorigenesis of NSCLC by transfecting recombinant retrovirus encoding shRNA targeting human CD59 into the human NSCLC cell line NCI-H157. CD59 expression in NSCLC was detected by immunocytochemistry (IHC). In the human NSCLC cell line NCI-H157, CD59 mRNA and protein expression suppressed with lentivirus-mediated RNAi was confirmed by using RT-PCR and western blotting, respectively. The proliferation and apoptosis of NCI-H157 cells was measured by using MTT assay and FACS. The resistance to complement cracking ability was detected by LDH assay. Caspase-3 expression in cells was assessed by IHC. Bcl-2 and Fas protein was determined by western blotting both in vitro and in vivo. CD59 is overexpressed in human NLCLC cancer. In NCI-H157 cells, lentivirus-mediated RNAi significantly reduced both CD59 mRNA and protein expression, which resulted in suppressing cell proliferation and increasing cell apoptosis. When incubated with fresh normal human serum (8%, v/v) for 1 h at 37˚C, the cell viability was decreased and cell apoptosis was increased in siCD59-infected NCI-H157 cells compared to siCD59-C-infected cells. Reduced CD59 expression led to increased expression of caspase-3 and Fas and decreased expression of Bcl-2. Furthermore, the nude mouse tumor graft weight was significantly decreased and survival rate was significantly increased in the siCD59 group. CD59 is overexpressed in human NLCLC. CD59 silencing in NSCLC cancer cells via retrovirus-mediated RNAi can enhance complement-mediated cell apoptosis, inhibiting the growth of NSCLC. CD59 may serve as a potential target for gene therapy in NSCLC.

Zhu J, Nie S, Wu J, Lubman DM
Target proteomic profiling of frozen pancreatic CD24+ adenocarcinoma tissues by immuno-laser capture microdissection and nano-LC-MS/MS.
J Proteome Res. 2013; 12(6):2791-804 [PubMed] Free Access to Full Article Related Publications
Cellular heterogeneity of solid tumors represents a common problem in mass spectrometry (MS)-based analysis of tissue specimens. Combining immuno-laser capture microdissection (iLCM) and mass spectrometry (MS) provides a means to study proteins that are specific for pure cell subpopulations in complex tissues. CD24, as a cell surface marker for detecting pancreatic cancer stem cells (CSCs), is directly correlated with the development and metastasis of pancreatic cancer. Herein, we describe an in-depth proteomic profiling of frozen pancreatic CD24(+) adenocarcinoma cells from early stage tumors using iLCM and LC-MS/MS and a comparison with CD24(-) cells dissected from patient-matched adjacent normal tissues. Approximately 40 nL of tissue was procured from each specimen and subjected to tandem MS analysis in triplicate. A total of 2665 proteins were identified, with 375 proteins in common that were significantly differentially expressed in CD24(+) versus CD24(-) cells by at least a 2-fold change. The major groups of the differentially overexpressed proteins are involved in promoting tumor cell migration and invasion, immune escape, and tumor progression. Three selected candidates relevant to mediating immune escape, CD59, CD70, and CD74, and a tumor promoter, TGFBI, were further validated by immunohistochemistry analysis on tissue microarrays. These proteins showed significantly increased expression in a large group of clinical pancreatic adenocarcinomas but were negative in all normal pancreas samples. The significant coexpression of these proteins with CD24 suggests that they may play important roles in the progression of pancreatic cancer and could serve as promising prognosis markers and novel therapeutic targets for this deadly disease.

Sahu B, Laakso M, Pihlajamaa P, et al.
FoxA1 specifies unique androgen and glucocorticoid receptor binding events in prostate cancer cells.
Cancer Res. 2013; 73(5):1570-80 [PubMed] Related Publications
The forkhead protein FoxA1 has functions other than a pioneer factor, in that its depletion brings about a significant redistribution in the androgen receptor (AR) and glucocorticoid receptor (GR) cistromes. In this study, we found a novel function for FoxA1 in defining the cell-type specificity of AR- and GR-binding events in a distinct fashion, namely, for AR in LNCaP-1F5 cells and for GR in VCaP cells. We also found different, cell-type and receptor-specific compilations of cis-elements enriched adjacent to the AR- and GR-binding sites. The AR pathway is central in prostate cancer biology, but the role of GR is poorly known. We find that AR and GR cistromes and transcription programs exhibit significant overlap, and GR regulates a large number of genes considered to be AR pathway-specific. This raises questions about the role of GR in maintaining the AR pathway under androgen-deprived conditions in castration-resistant prostate cancer patients. However, in the presence of androgen, ligand-occupied GR acts as a partial antiandrogen and attenuates the AR-dependent transcription program. .

Kohler H, Rector K, Amick J
Generation of a potent recombinant homophilic chimeric anti-CD20 antibody.
Hybridoma (Larchmt). 2012; 31(6):395-402 [PubMed] Related Publications
Previously we increased the potency of therapeutic antibodies in targeting, induction of apoptosis, and growth inhibition in vitro and in vivo by chemically conjugating a homophilic peptide to the antibody. Here, we describe the construction of a chimeric fusion gene derived from the murine anti-CD20 antibody (1F5) variable region, with an engineered homophilic domain at the C-terminus of the human IgG1 sequence. The construct was expressed in CHO suspension cells and purified. The potency of the homophilic anti-CD20 antibody was compared to a chimeric antibody without the engineered homophilic domain. In this comparison, the homophilic anti-CD20 antibody showed increased binding to a human CD20 cell line, and significantly more ADCC, CDC, and induction of apoptosis in three cell lines. In addition, the homophilic anti-CD20 antibody demonstrated increased inhibition of proliferation of two cell lines. These data show that homophilic fusion protein antibodies with enhanced therapeutic potency can be produced with industry-standard fermentation protocols.

Terp MG, Lund RR, Jensen ON, et al.
Identification of markers associated with highly aggressive metastatic phenotypes using quantitative comparative proteomics.
Cancer Genomics Proteomics. 2012 Sep-Oct; 9(5):265-73 [PubMed] Related Publications
BACKGROUND: The spread of cancer cells from a primary tumor to form metastases at distant sites is a complex process that remains poorly defined. Certain tumor cells are more aggressive and thus lead to rapid development of multiple distant metastases. Here, we identify proteins associated with these aggressive phenotypes.
MATERIALS AND METHODS: To identify proteins associated with cancer cell aggressiveness, we used comparative, quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteome analysis of a unique metastasis model comprised of three isogenic human breast cancer cell lines that are equally tumorigenic in mice, but display different metastatic potentials ranging from non-metastatic, intermediate-metastatic and highly-metastatic. The altered expression of selected proteins was subsequently confirmed by immunocyto- and immunohistochemistry.
RESULTS: The difference in metastatic capabilities was initially confirmed using live animal imaging. Comparative, quantitative proteomics identified 414 proteins, out of which 44 exhibited altered expression between the metastatic and non-metastatic cell lines. The proteins correlating with the aggressiveness of metastasis included leucine-rich repeat containing 59 (LRRC59), while CD59 and chondroitin sulfate proteoglycan 4 (CSPG4) exhibited an inverse correlation with metastatic capability. The altered expression levels of these proteins were biochemically confirmed, as well as demonstrated in xenografts generated from these cell lines. This analysis further demonstrated that the three proteins were associated with the aggressiveness of metastasis rather than metastasis colonization per se.
CONCLUSION: Our study provides novel insights into key proteins associated with the metastatic potential of breast cancer cells and identified LRRC59, CD59 and CSPG4 as candidates that merit further study.

Bellone S, Roque D, Cocco E, et al.
Downregulation of membrane complement inhibitors CD55 and CD59 by siRNA sensitises uterine serous carcinoma overexpressing Her2/neu to complement and antibody-dependent cell cytotoxicity in vitro: implications for trastuzumab-based immunotherapy.
Br J Cancer. 2012; 106(9):1543-50 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: We evaluated the expression of CD46, CD55 and CD59 membrane-bound complement-regulatory proteins (mCRPs) in primary uterine serous carcinoma (USC) and the ability of small interfering RNA (siRNA) against these mCRPs to sensitise USC to complement-dependent cytotoxicity (CDC) and antibody (trastuzumab)-dependent cellular cytotoxicity (ADCC) in vitro.
METHODS: Membrane-bound complement-regulatory proteins expression was evaluated using real-time PCR (RT-PCR) and flow cytometry, whereas Her2/neu expression and c-erbB2 gene amplification were assessed using immunohistochemistry, flow cytometry and fluorescent in-situ hybridisation. The biological effect of siRNA-mediated knockdown of mCRPs on HER2/neu-overexpressing USC cell lines was evaluated in CDC and ADCC 4-h chromium-release assays.
RESULTS: High expression of mCRPs was found in USC cell lines when compared with normal endometrial cells (P<0.05). RT-PCR and FACS analyses demonstrated that anti-mCRP siRNAs were effective in reducing CD46, CD55 and CD59 expression on USC (P<0.05). Baseline complement-dependent cytotoxicity (CDC) against USC cell lines was low (mean ± s.e.m.=6.8 ± 0.9%) but significantly increased upon CD55 and CD59 knockdown (11.6 ± 0.8% and 10.7 ± 0.9%, respectively, P<0.05). Importantly, in the absence of complement, both CD55 and CD59, but not CD46, knockdowns significantly augmented ADCC against USC overexpressing Her2/neu.
CONCLUSION: Uterine serous carcinoma express high levels of the mCRPs CD46, CD55 and CD59. Small interfering RNA inhibition of CD55 and CD59, but not CD46, sensitises USC to both CDC and ADCC in vitro, and if specifically targeted to tumour cells, may significantly increase trastuzumab-mediated therapeutic effect in vivo.

Ge X, Wang IN, Toma I, et al.
Human amniotic mesenchymal stem cell-derived induced pluripotent stem cells may generate a universal source of cardiac cells.
Stem Cells Dev. 2012; 21(15):2798-808 [PubMed] Free Access to Full Article Related Publications
Human amniotic mesenchymal stem cells (hAMSCs) demonstrated partially pluripotent characteristics with a strong expression of Oct4 and Nanog genes and immunomodulatory properties characterized by the absence of HLA-DR and the presence of HLA-G and CD59. The hAMSCs were reprogrammed into induced pluripotent stem cells (iPSCs) that generate a promising source of universal cardiac cells. The hAMSC-derived iPSCs (MiPSCs) successfully underwent robust cardiac differentiation to generate cardiomyocytes. This study investigated 3 key properties of the hAMSCs and MiPSCs: (1) the reprogramming efficiency of the partially pluripotent hAMSCs to generate MiPSCs; (2) immunomodulatory properties of the hAMSCs and MiPSCs; and (3) the cardiac differentiation potential of the MiPSCs. The characteristic iPSC colony formation was observed within 10 days after the transduction of the hAMSCs with a single integration polycistronic vector containing 4 Yamanaka factors. Immunohistology and reverse transcription-polymerase chain reaction assays revealed that the MiPSCs expressed stem cell surface markers and pluripotency-specific genes. Furthermore, the hAMSCs and MiPSCs demonstrated immunomodulatory properties enabling successful engraftment in the SVJ mice. Finally, the cardiac differentiation of MiPSCs exhibited robust spontaneous contractility, characteristic calcium transience across the membrane, a high expression of cardiac genes and mature cardiac phenotypes, and a contractile force comparable to cardiomyocytes. Our results demonstrated that the hAMSCs are reprogrammed with a high efficiency into MiPSCs, which possess pluripotent, immunomodulatory, and precardiac properties. The MiPSC-derived cardiac cells express a c-kit cell surface marker, which may be employed to purify the cardiac cell population and enable allogeneic cardiac stem cell therapy.

Guzzo F, Bellone S, Buza N, et al.
HER2/neu as a potential target for immunotherapy in gynecologic carcinosarcomas.
Int J Gynecol Pathol. 2012; 31(3):211-21 [PubMed] Free Access to Full Article Related Publications
Carcinosarcomas of the female genital tract are rare tumors with an aggressive clinical behavior. Trastuzumab, a humanized monoclonal antibody, acts by binding to HER2/neu extracellular domain and exhibits therapeutic efficacy in HER2/neu-overexpressing cancers. Two uterine carcinosarcomas (UMMT-ARK-1, UMMT-ARK-2) and 2 ovarian carcinosarcomas (OMMT-ARK-1, OMMT-ARK-2) were established as primary tumor cell lines in vitro and evaluated for HER2/neu expression by immunohistochemistry, fluorescent in situ hybridization analysis, quantitative real-time polymerase chain reaction, and for membrane-bound complement regulatory proteins CD46, CD55, and CD59 by flow cytometry. Sensitivity to trastuzumab-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity was studied in 5-hr chromium release assays. HER2/neu expression was demonstrated in OMMT-ARK-1 and OMMT-ARK-2. OMMT-ARK-2 demonstrated an amplification of the c-erbB2 gene by fluorescent in situ hybridization analysis and a high sensitivity to ADCC (mean killing, 45.6%; range, 32.3%-72.6%). A lower level of killing was detected against the fluorescent in situ hybridization analysis-negative OMMT-ARK-1 cell line (mean, 26.5%; range, 21.0%-31.8%). CD46, CD55, and CD59 membrane-bound complement regulatory proteins were expressed at high levels in all primary mixed müllerian tumor cell lines, and all these tumors were found to be highly resistant to complement-dependent cytotoxicity with or without trastuzumab. Addition of untreated and heat-inactivated plasma did not significantly decrease ADCC against OMMT-ARK-2 cell line, suggesting that while the cell line is highly resistant to complement, irrelevant IgG does not significantly alter the ability of trastuzumab to mediate ADCC. Our results suggest that HER2/neu may represent a novel target for the immunotherapy of a subset of human carcinosarcomas refractory to salvage chemotherapy.

Xie XH, Gao MH, Zhang B, et al.
Post-transcriptional CD59 gene silencing by siRNAs induces enhanced human T lymphocyte response to tumor cell lysate-loaded DCs.
Cell Immunol. 2012; 274(1-2):1-11 [PubMed] Related Publications
CD59 is a complement regulatory protein known to prevent the membrane attack complex (MAC) from assembling. To investigate the role of CD59 molecules in human T cell activation in response to exogenous antigens, gene silencing via small interfering RNAs (siRNAs) was carried out. Subsequent T cell activation in response to both autologous dendritic cells (DCs) loaded with tumor lysate and beads coated with anti-CD3, anti-CD28 and anti-CD59 antibodies was investigated. The findings demonstrated that decreased CD59 expression on T cells significantly enhanced activation and proliferation of CD4(+) T cells and CD8(+) T cells while the expansion of CD4(+) CD25(+) regulatory T cells (Tregs) was not affected, and CD59 mediated inhibition of T cell activation requires the binding of CD59 with its ligand on antigen-presenting cells (APCs). The data support that CD59 down-regulates antigen-specific activation of human T lymphocytes in a ligand-dependent manner.

Li B, Chu X, Gao M, Xu Y
The effects of CD59 gene as a target gene on breast cancer cells.
Cell Immunol. 2011; 272(1):61-70 [PubMed] Related Publications
The retroviral-vector-targeted CD59 gene (pSUPER-siCD59) was constructed and transfected into breast cells (MCF-7). The results demonstrated that the retroviral vector-mediated RNAi successfully suppressed human CD59 gene. The expression of CD59 decreased at both mRNA and protein levels. Knockdown of CD59 abrogated its protective effect on complement-mediated cytolysis. Fas and caspase-3 were remarkably upregulated, which induced apoptosis and tumor growth suppression in MCF-7 cells. In addition, overexpression of CD59 promoted the proliferation of MCF-7 cells and inhibited anti-apoptotic Bcl-2 expression. In conclusion, CD59 may be a promising target in the gene therapy of breast cancer.

Sahu B, Laakso M, Ovaska K, et al.
Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer.
EMBO J. 2011; 30(19):3962-76 [PubMed] Free Access to Full Article Related Publications
High androgen receptor (AR) level in primary tumour predicts increased prostate cancer-specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR-binding sites (ARBs) on LNCaP-1F5 cell chromatin that was commensurate with changes in androgen-dependent gene expression signature. We identified three distinct classes of ARBs and androgen-responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid-dependent signalling in LNCaP-1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.

Cocco E, Varughese J, Buza N, et al.
Tissue factor expression in ovarian cancer: implications for immunotherapy with hI-con1, a factor VII-IgGF(c) chimeric protein targeting tissue factor.
Clin Exp Metastasis. 2011; 28(7):689-700 [PubMed] Free Access to Full Article Related Publications
We evaluated the expression of tissue factor (TF) in ovarian cancer (EOC) and the potential of hI-con1, an antibody-like molecule targeting TF, as a novel form of therapy against chemotherapy-resistant ovarian disease. We studied the expression of TF in 88 EOC by immunohistochemistry (IHC) and real-time-PCR (qRT-PCR) and the levels of membrane-bound-complement-regulatory-proteins CD46, CD55 and CD59 in primary EOC cell lines by flow-cytometry. Sensitivity to hI-con1-dependent-cell-mediated-cytotoxicity (IDCC), complement-dependent-cell-cytotoxicity and inhibition of IDCC by γ-immunoglobulin were evaluated in 5-h (51)chromium-release-assays. Cytoplasmic and/or membrane TF expression was observed in 24 out of 25 (96%) of the EOC samples tested by IHC, but not in normal ovarian-tissue. EOC with clear cell histology significantly overexpress TF when compared to serous, endometrioid, or undifferentiated tumors by qRT-PCR. With a single exception, all primary EOC that overexpressed TF demonstrated high levels of CD46, CD55 and CD59 and regardless of their histology or resistance to chemotherapy, were highly sensitive to IDCC. The effect of complement and physiologic doses of γ-immunoglobulin on IDCC in ovarian cancer cell lines overexpressing TF was tumor specific and related to the overexpression of CD59 on tumor cells. Small-interfering-RNA-mediated knockdown of CD59 expression in ovarian tumors significantly increased hI-con1-mediated cytotoxic activity in vitro. Finally, low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (P < 0.01). hI-con1 molecule induces strong cytotoxicity against primary chemotherapy-resistant ovarian cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of ovarian tumors refractory to standard treatment modalities.

Shan C, Zhang S, Cui W, et al.
Hepatitis B virus X protein activates CD59 involving DNA binding and let-7i in protection of hepatoma and hepatic cells from complement attack.
Carcinogenesis. 2011; 32(8):1190-7 [PubMed] Related Publications
Emerging evidence has shown that hepatitis B virus (HBV) X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma. Complement regulatory proteins including CD46, CD55 and CD59 contribute to escape of tumor cells from complement-dependent cytotoxicity (CDC). However, little is known about the potential role of HBx in anti-CDC activity during hepatocarcinogenesis. In the present study, we for the first time report that HBx decreases the sensitivity of hepatoma and hepatic cells to CDC. Coincidentally, we demonstrated that HBx increased the promoter activity of CD59, as well as their messenger RNA and protein levels. Moreover, flow cytometry showed the increased expression level of CD59 protein on the surface of HBx-positive cells. Of interest, we found that HBx up-regulated CD59 by binding with cAMP response element-binding to the promoter region of the CD59 gene using chromatin immunoprecipitation assay. In addition, we showed that HBx up-regulated CD59 by let-7i at post-transcriptional regulation level. Our data showed that the deposition of C5b-9 were decreased on the cell surface in HepG2-X cells relative to HepG2 cells, suggesting that increased CD59 mediated by HBx prevents the formation of functional membrane attack complex. Furthermore, we demonstrated that down-regulation of CD59 was sufficient to abolish the resistance capability of CDC in HBx-positive cells by RNA interference (siRNA) in vitro and in vivo. Thus, we conclude that HBx contributes to cells resistance to CDC through CD59. Therapeutically, CD59 may serve as a target in HBV-associated hepatoma patients.

Geis N, Zell S, Rutz R, et al.
Inhibition of membrane complement inhibitor expression (CD46, CD55, CD59) by siRNA sensitizes tumor cells to complement attack in vitro.
Curr Cancer Drug Targets. 2010; 10(8):922-31 [PubMed] Related Publications
The efficacy of cancer-immunotherapy with complement-activating monoclonal antibodies is limited by over-expression of one or more membrane-bound complement regulatory proteins (mCRPs: CD46, CD55, CD59) on the surface of neoplastic cells. In this study we designed small interfering RNAs (siRNAs) for posttranscriptional gene knock down of CD46, CD55 and CD59 aiming at to sensitize tumor cells to complement attack and thereby to better exploit complement for tumor cell destruction. Tumor cell lines of different origin, such as Du145 (prostate), BT474 (breast) and K562 (erythroleukemia) were selected for the study. FACS-analysis demonstrated that siRNA anti-CD46(301) reduced CD46 protein expression up to 80%, siRNA anti-CD55(255) diminished CD55 protein expression up to 49%, and CD59 protein expression was inhibited up to 82% by siRNA anti-CD59(1339). Time course experiments revealed a long-lasting silencing effect with >50% complement regulator inhibition up to day 13. Upon mCRP knock down, complement-dependent cytotoxicity (CDC) was augmented by 20-30% for CD46, by up to 24% for CD55 and by up to 55% for CD59. The combined inhibition of all three inhibitors further enhanced CDC by up to 66%. Dependent on the cell line, CD46 and CD55 downregulation increased significantly C3 ospsonization, which is known to support cell-mediated defense mechanisms. mCRP blocking antibodies were only partly able to further augment the tumor cells' susceptibility to complement lysis. Thus, siRNA-induced inhibition of complement regulator expression clearly sensitizes malignant cells to complement attack and, if specifically targeted to the tumor, appears suited as adjuvant to improve antibody-based cancer immunotherapy.

Kolev MV, Ruseva MM, Morgan BP, Donev RM
Targeting neural-restrictive silencer factor sensitizes tumor cells to antibody-based cancer immunotherapy in vitro via multiple mechanisms.
J Immunol. 2010; 184(11):6035-42 [PubMed] Related Publications
Tumor cells escape clearance by complement by abundantly expressing CD59 and other membrane complement regulators. Recently, we designed a peptide derived from the neural-restrictive silencer factor (REST), REST68, which we showed to inhibit expression of CD59 in tumors lacking the full-length REST and proposed a detailed model for regulation of CD59 expression via interplay between REST and nucleolin (NCL) transcription factors. In this paper, we study in detail the mechanisms for sensitization of malignant cells to Ab-based cancer immunotherapy by the REST68 peptide and the implications of the REST/NCL model for the design of treatment resulting in higher tumor susceptibility. REST68 inhibited CD59 expression in malignant cells expressing either truncated or full-length REST, but not in nonmalignant cells. However, activation of protein kinase C (PKC) in nonmalignant cells, a process that contributes to cellular transformation, phosphorylated NCL and enabled suppression of CD59 expression by the REST68. Combined treatment of different tumor types with REST68 and PKC inhibitor synergized to further suppress CD59 expression and reduce resistance to complement lysis. The combined treatment also increased susceptibility of tumors expressing either of the REST isoforms to PBMC-mediated killing, which, at least in part, accounted for the strong promotion of apoptosis by the REST68/PKC inhibitor. These data demonstrate that REST68 sensitizes tumors to Ab-based cancer immunotherapy via multiple mechanisms. Furthermore, the REST/NCL interplay model for regulation of expression of cd59 and other genes involved in cell survival enables the design of treatments for different tumor types to achieve more efficient tumor clearance.

Tediose T, Kolev M, Sivasankar B, et al.
Interplay between REST and nucleolin transcription factors: a key mechanism in the overexpression of genes upon increased phosphorylation.
Nucleic Acids Res. 2010; 38(9):2799-812 [PubMed] Free Access to Full Article Related Publications
Non-malignant cells can be transformed via the activation of kinases that control degradation of neural-restrictive silencer factor (REST). Here, we identify a mechanism that contributes to the activation of genes, expression of which is controlled by responsive elements containing overlapping binding sites for REST and nucleolin. We demonstrate that both phosphorylated and non-phosphorylated nucleolin-bound DNA; however, only phosphorylated nucleolin successfully competed with either full-length REST or a REST-derived DNA-binding peptide, REST68, for binding to the overlapping binding sites. We show that this interplay between the two transcription factors regulates the activation of cell survival and immunomodulatory genes in tumors and non-malignant cells with activated protein kinase C, which is accompanied with alterations in cell proliferation and apoptosis. We propose a model for the regulation of these genes, which brings a new insight into the molecular mechanisms that control cellular transformation driven by activation of protein kinases.

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