DNA Methylation of the ABO Promoter Underlies Loss of ABO Allelic Expression in a Significant Proportion of Leukemic Patients

Background: Loss of A, B and H antigens from the red blood cells of patients with myeloid malignancies is a frequent occurrence. Previously, we have reported alterations in ABH antigens on the red blood cells of 55% of patients with myeloid malignancies. Methodology/Principal Findings: To determine the underlying molecular mechanisms of this loss, we assessed ABO allelic expression in 21 patients with ABH antigen loss previously identified by flow cytometric analysis as well as an additional 7 patients detected with ABH antigen changes by serology. When assessing ABO mRNA allelic expression, 6/12 (50%) patients with ABH antigen loss detected by flow cytometry and 5/7 (71%) of the patients with ABH antigen loss detected by serology had a corresponding ABO mRNA allelic loss of expression. We examined the ABO locus for copy number and DNA methylation alterations in 21 patients, 11 with loss of expression of one or both ABO alleles, and 10 patients with no detectable allelic loss of ABO mRNA expression. No loss of heterozygosity (LOH) at the ABO locus was observed in these patients. However in 8/11 (73%) patients with loss of ABO allelic expression, the ABO promoter was methylated compared with 2/10 (20%) of patients with no ABO allelic expression loss (P = 0.03). Conclusions/Significance: We have found that loss of ABH antigens in patients with hematological malignancies is associated with a corresponding loss of ABO allelic expression in a significant proportion of patients. Loss of ABO allelic expression was strongly associated with DNA methylation of the ABO promoter.Tina Bianco-Miotto, Damian J. Hussey, Tanya K. Day, Denise S. O'Keefe and Alexander Dobrovi

Genetic Deletion of the Desmosomal Component Desmoplakin Promotes Tumor Microinvasion in a Mouse Model of Pancreatic Neuroendocrine Carcinogenesis

We used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to profile the transcriptome of pancreatic neuroendocrine tumors (PNET) that were either non-invasive or highly invasive, seeking to identify pro- and anti-invasive molecules. Expression of multiple components of desmosomes, structures that help maintain cellular adhesion, was significantly reduced in invasive carcinomas. Genetic deletion of one of these desmosomal components, desmoplakin, resulted in increased local tumor invasion without affecting tumor growth parameters in RT2 PNETs. Expression of cadherin 1, a component of the adherens junction adhesion complex, was maintained in these tumors despite the genetic deletion of desmoplakin. Our results demonstrate that loss of desmoplakin expression and resultant disruption of desmosomal adhesion can promote increased local tumor invasion independent of adherens junction status

Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition

INTRODUCTION: This report describes the isolation and characterization of three new murine mammary epithelial cell lines derived from mammary tumors from MMTV (mouse mammary tumor virus)/activated Neu + TβRII-AS (transforming growth factor [TGF]-β type II receptor antisense RNA) bigenic mice (BRI-JM01 and BRI-JM05 cell lines) and MMTV/activated Neu transgenic mice (BRI-JM04 cell line). METHODS: The BRI-JM01, BRI-JM04, and BRI-JM05 cell lines were analyzed for transgene expression, their general growth characteristics, and their sensitivities to several growth factors from the epidermal growth factor (EGF) and TGF-β families (recombinant human EGF, heregulin-β(1 )and TGF-β(1)). The BRI-JM01 cells were observed to undergo a striking morphologic change in response to TGF-β(1), and they were therefore further investigated for their ability to undergo a TGF-β-induced epithelial-to-mesenchymal transition (EMT) using motility assays and immunofluorescence microscopy. RESULTS: We found that two of the three cell lines (BRI-JM04 and BRI-JM05) express the Neu transgene, whereas, unexpectedly, both of the cell lines that were established from MMTV/activated Neu + TβRII-AS bigenic tumors (BRI-JM01 and BRI-JM05) do not express the TβRII-AS transgene. The cuboidal BRI-JM01 cells exhibit a short doubling time and are able to form confluent monolayers. The BRI-JM04 and BRI-JM05 cell lines are morphologically much less uniform, grow at a much slower rate, and do not form confluent monolayers. Only the BRI-JM05 cells can form colonies in soft agar. In contrast, all three cell lines form colonies in Matrigel, although the BRI-JM04 and BRI-JM05 cell lines do so more efficiently than the BRI-JM01 cell line. All three cell lines express the cell surface marker E-cadherin, confirming their epithelial character. Proliferation assays showed that the three cell lines respond differently to recombinant human EGF and heregulin-β(1), and that all are growth inhibited by TGF-β(1), but that only the BRI-JM01 cell line undergoes an EMT and exhibits increased motility upon TGF-β(1 )treatment. CONCLUSION: We suggest that the BRI-JM04 and BRI-JM05 cell lines can be used to investigate Neu oncogene driven mammary tumorigenesis, whereas the BRI-JM01 cell line will be useful for studying TGF-β(1)-induced EMT

Clinical and immunological evaluation of anti-apoptosis protein, survivin-derived peptide vaccine in phase I clinical study for patients with advanced or recurrent breast cancer

<p>Abstract</p> <p>Background</p> <p>We previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to <b>c</b>linically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer.</p> <p>Methods</p> <p>We set up two protocols. In the first protocol, 10 patients were vaccinated with escalating doses (0.1–1.0 mg) of survivin-2B peptide alone 4 times every 2 weeks. In the second protocol, 4 patients were vaccinated with the peptide at a dose of 1.0 mg mixed with IFA 4 times every 2 weeks.</p> <p>Results</p> <p>In the first protocol, no adverse events were observed during or after vaccination. In the second protocol, two patients had induration at the injection site. One patient had general malaise (grade 1), and another had general malaise (grade 1) and fever (grade 1). Peptide vaccination was well tolerated in all patients. In the first protocol, tumor marker levels increased in 8 patients, slightly decreased in 1 patient and were within the normal range during this clinical trial in 1 patient. With regard to tumor size, two patients were considered to have stable disease (SD). Immunologically, in 3 of the 10 patients (30%), an increase of the peptide-specific CTL frequency was detected. In the second protocol, an increase of the peptide-specific CTL frequency was detected in all 4 patients (100%), although there were no significant beneficial clinical responses. ELISPOT assay showed peptide-specific IFN-γ responses in 2 patients in whom the peptide-specific CTL frequency in tetramer staining also was increased in both protocols.</p> <p>Conclusion</p> <p>This phase I clinical study revealed that survivin-2B peptide vaccination was well tolerated. The vaccination with survivin-2B peptide mixed with IFA increased the frequency of peptide-specific CTL more effectively than vaccination with the peptide alone, although neither vaccination could induce efficient clinical responses. Considering the above, the addition of another effectual adjuvant such as a cytokine, heat shock protein, etc. to the vaccination with survivin-2B peptide mixed with IFA might induce improved immunological and clinical responses.</p

The E-cadherin repressor slug and progression of human extrahepatic hilar cholangiocarcinoma

<p>Abstract</p> <p>Objectives</p> <p>This study explored the expression and function of Slug in human extrahepatic hilar cholangiocarcinoma (EHC) to identify its role in tumor progression.</p> <p>Methods</p> <p>The expression of Snail and Slug mRNA in 52 human tissue samples of EHC was investigated. The mRNA of Snail and Slug were quantified using reverse transcriptase-PCR, and correlations with E-cadherin expression and clinicopathological factors were investigated. We then investigated transfection of Slug cDNA in endogenous E-cadherin-positive human EHC FRH0201 cells, selectively induced the loss of E-cadherin protein expression, and then small interfering RNA (siRNA) for inhibition of Slug expression in endogenous Slug-positive human EHC QBC939 cells, selectively induced the loss of Slug protein expression. A Boyden chamber transwell assay was used for invasion.</p> <p>Results</p> <p>Slug mRNA was overexpressed in 18 cases (34.6%) of EHC compared with adjacent noncancerous tissue. E-Cadherin protein expression determined in the same 52 cases by immunohistochemistry was significantly down-regulated in those cases with Slug mRNA overexpression (P = 0.0001). The tumor and nontumor ratio of Slug mRNA was correlated with nodal metastasis(p = 0.0102), distant metastasis (p = 0.0001)and Survival time(p = 0.0443). However, Snail mRNA correlated with neither E-cadherin expression nor tumor invasiveness. By inhibiting Slug expression by RNA interference, we found that reduced Slug levels upregulated E-cadherin and decreased invasion in QBC939 cell. When the QBC939 cells was infected with Slug cDNA,, significant E-cadherin was downregulated and increased invasion in QBC939 cell.</p> <p>Conclusions</p> <p>The results suggested that Slug expression plays an important role in both the regulation of E-cadherin expression and in the acquisition of invasive potential in human EHC. Slug is possibly a potential target for an antitumor therapy blocking the functions of invasion and metastasis in human EHCs.</p

Gain in cellular organization of inflammatory breast cancer: A 3D in vitro model that mimics the in vivo metastasis

<p>Abstract</p> <p>Background</p> <p>The initial step of metastasis in carcinomas, often referred to as the epithelial-mesenchymal transition (EMT), occurs via the loss of adherens junctions (e.g. cadherins) by the tumor embolus. This leads to a subsequent loss of cell polarity and cellular differentiation and organization, enabling cells of the embolus to become motile and invasive. However highly malignant inflammatory breast cancer (IBC) over-expresses E-cadherin. The human xenograft model of IBC (MARY-X), like IBC, displays the signature phenotype of an exaggerated degree of lymphovascular invasion (LVI) <it>in situ </it>by tumor emboli. An intact E-cadherin/α, β-catenin axis mediates the tight, compact clump of cells found both <it>in vitro </it>and <it>in vivo </it>as spheroids and tumor emboli, respectively.</p> <p>Methods</p> <p>Using electron microscopy and focused ion beam milling to acquire <it>in situ </it>sections, we performed ultrastructural analysis of both an IBC and non-IBC, E-cadherin positive cell line to determine if retention of this adhesion molecule contributed to cellular organization.</p> <p>Results</p> <p>Here we report through ultrastructural analysis that IBC exhibits a high degree of cellular organization with polar elements such as apical/lateral positioning of E-cadherin, apical surface microvilli, and tortuous lumen-like (canalis) structures. In contrast, agarose-induced spheroids of MCF-7, a weakly invasive E-cadherin positive breast carcinoma cell line, do not exhibit ultrastructural polar features.</p> <p>Conclusions</p> <p>This study has determined that the highly metastatic IBC with an exaggerated malignant phenotype challenges conventional wisdom in that instead of displaying a loss of cellular organization, IBC acquires a highly structured architecture.</p> <p>These findings suggest that the metastatic efficiency might be linked to the formation and maintenance of these architectural features. The comparative architectural features of both the spheroid and embolus of MARY-X provide an <it>in vitro </it>model with tractable <it>in vivo </it>applications.</p

Cdc6 expression represses E-cadherin transcription and activates adjacent replication origins

The Cdc6 replication licensing factor acts as a molecular switch at the E-cadherin locus, leading to E-cadherin transcriptional repression and local activation of replication

Effect of ABCB1 and ABCC3 Polymorphisms on Osteosarcoma Survival after Chemotherapy: A Pharmacogenetic Study

Background: Standard treatment for osteosarcoma patients consists of a combination of cisplatin, adriamycin, and methotrexate before surgical resection of the primary tumour, followed by postoperative chemotherapy including vincristine and cyclophosphamide. Unfortunately, many patients still relapse or suffer adverse events. We examined whether common germline polymorphisms in chemotherapeutic transporter and metabolic pathway genes of the drugs used in standard osteosarcoma treatment may predict treatment response. Methodology/Principal Findings: In this study we screened 102 osteosarcoma patients for 346 Single Nucleotide Polymorphisms (SNPs) and 2 Copy Number Variants (CNVs) in 24 genes involved in the metabolism or transport of cisplatin, adriamycin, methotrexate, vincristine, and cyclophosphamide. We studied the association of the genotypes with tumour response and overall survival. We found that four SNPs in two ATP-binding cassette genes were significantly associated with overall survival: rs4148416 in ABCC3 (per-allele HR = 8.14, 95%CI = 2.73-20.2, p-value = 5.1×10 -5), and three SNPs in ABCB1, rs4148737 (per-allele HR = 3.66, 95%CI = 1.85-6.11, p-value = 6.9×10 -5), rs1128503 and rs10276036 (r 2 = 1, per-allele HR = 0.24, 95%CI = 0.11-0.47 p-value = 7.9×10 -5). Associations with these SNPs remained statistically significant after correction for multiple testing (all corrected p-values [permutation test] ≤0.03). Conclusions: Our findings suggest that these polymorphisms may affect osteosarcoma treatment efficacy. If these associations are independently validated, these variants could be used as genetic predictors of clinical outcome in the treatment of osteosarcoma, helping in the design of individualized therapyThis work was supported by the AECC (Asociación Española contra el Cáncer), FIS (Fondo de Investigación Sanitaria-Instituto de Salud Carlos III) and the ‘‘Inocente Inocente’’ Foundatio

CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer

BACKGROUND: The E-cadherin gene (CDH1) maps, at chromosome 16q22.1, a region often associated with loss of heterozygosity (LOH) in human breast cancer. LOH at this site is thought to lead to loss of function of this tumor suppressor gene and was correlated with decreased disease-free survival, poor prognosis, and metastasis. Differential CpG island methylation in the promoter region of the CDH1 gene might be an alternative way for the loss of expression and function of E-cadherin, leading to loss of tissue integrity, an essential step in tumor progression. METHODS: The aim of our study was to assess, by Methylation-Specific Polymerase Chain Reaction (MSP), the methylation pattern of the CDH1 gene and its possible correlation with the expression of E-cadherin and other standard immunohistochemical parameters (Her-2, ER, PgR, p53, and K-67) in a series of 79 primary breast cancers (71 infiltrating ductal, 5 infiltrating lobular, 1 metaplastic, 1 apocrine, and 1 papillary carcinoma). RESULTS: CDH1 hypermethylation was observed in 72% of the cases including 52/71 ductal, 4/5 lobular carcinomas and 1 apocrine carcinoma. Reduced levels of E-cadherin protein were observed in 85% of our samples. Although not statistically significant, the levels of E-cadherin expression tended to diminish with the CDH1 promoter region methylation. In the group of 71 ductal cancinomas, most of the cases of showing CDH1 hypermethylation also presented reduced levels of expression of ER and PgR proteins, and a possible association was observed between CDH1 methylation and ER expression (p = 0.0301, Fisher's exact test). However, this finding was not considered significant after Bonferroni correction of p-value. CONCLUSION: Our preliminary findings suggested that abnormal CDH1 methylation occurs in high frequencies in infiltrating breast cancers associated with a decrease in E-cadherin expression in a subgroup of cases characterized by loss of expression of other important genes to the mammary carcinogenesis process, probably due to the disruption of the mechanism of maintenance of DNA methylation in tumoral cells

Xanthene Food Dye, as a Modulator of Alzheimer's Disease Amyloid-beta Peptide Aggregation and the Associated Impaired Neuronal Cell Function

Alzheimer's disease (AD) is the most common form of dementia. AD is a degenerative brain disorder that causes problems with memory, thinking and behavior. It has been suggested that aggregation of amyloid-beta peptide (Aβ) is closely linked to the development of AD pathology. In the search for safe, effective modulators, we evaluated the modulating capabilities of erythrosine B (ER), a Food and Drug Administration (FDA)-approved red food dye, on Aβ aggregation and Aβ-associated impaired neuronal cell function.In order to evaluate the modulating ability of ER on Aβ aggregation, we employed transmission electron microscopy (TEM), thioflavin T (ThT) fluorescence assay, and immunoassays using Aβ-specific antibodies. TEM images and ThT fluorescence of Aβ samples indicate that protofibrils are predominantly generated and persist for at least 3 days. The average length of the ER-induced protofibrils is inversely proportional to the concentration of ER above the stoichiometric concentration of Aβ monomers. Immunoassay results using Aβ-specific antibodies suggest that ER binds to the N-terminus of Aβ and inhibits amyloid fibril formation. In order to evaluate Aβ-associated toxicity we determined the reducing activity of SH-SY5Y neuroblastoma cells treated with Aβ aggregates formed in the absence or in the presence of ER. As the concentration of ER increased above the stoichiometric concentration of Aβ, cellular reducing activity increased and Aβ-associated reducing activity loss was negligible at 500 µM ER.Our findings show that ER is a novel modulator of Aβ aggregation and reduces Aβ-associated impaired cell function. Our findings also suggest that xanthene dye can be a new type of small molecule modulator of Aβ aggregation. With demonstrated safety profiles and blood-brain permeability, ER represents a particularly attractive aggregation modulator for amyloidogenic proteins associated with neurodegenerative diseases