Epigenetic inactivation of the MIR34B/C in multiple myeloma

Abstract We postulated that MIR34B/C, a direct transcriptional target of TP53, might be inactivated by promoter hypermethylation in multiple myeloma (MM). MIR34B/C promoter methylation was studied in 8 normal marrow controls, 8 MM cell lines, 95 diagnostic, and 23 relapsed/progressed MM samples by methylation-specific PCR. MIR34B/C was methylated in 6 (75.0%) MM cell lines but not normal controls. 5-Aza-2′-deoxycytidine led to MIR34B/C promoter demethylation and MIR34B reexpression. Moreover, restoration of MIR34B led to reduced cellular proliferation and enhanced apoptosis of myeloma cells. In primary samples, methylation of MIR34B/C occurred in 5.3% at diagnosis and 52.2% at relapse/disease progression (P < .001). In 12 MM patients with paired samples at diagnosis and relapse/progression, MIR34B/C methylation was acquired in 6 at relapse/progression. In conclusion, MIR34B/C is a tumor suppressor in myeloma. Hypermethylation of MIR34B/C is tumor-specific. Frequent MIR34B/C hypermethylation during relapse/progression but not at diagnosis implicated a role of MIR34B/C hypermethylation in myeloma relapse/progression.

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Molecular Basis of Genomic Instability and Progression in Multiple Myeloma: Potential Role of Apurinic (Apyrimidinic) Endonuclease.

Blood ◽

10.1182/blood.v106.11.1561.1561 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1561-1561

Author(s):

Masood A. Shammas ◽

Hemanta Koley ◽

Paola Neri ◽

Pierfrancesco Tassone ◽

Ramesh B. Batchu ◽

...

Keyword(s):

Multiple Myeloma ◽

Genomic Instability ◽

Cell Lines ◽

Molecular Basis ◽

Endonuclease Activity ◽

Ap Endonuclease ◽

Myeloma Cells ◽

Genome Wide ◽

New Mutations

Abstract Genetic instability is a prominent feature of most cancers including multiple myeloma (MM) and is responsible for ongoing accrual of mutational changes which may lead to development of drug resistance and metastasis. The molecular basis for the generation of genetic diversity in MM is therefore extremely important to understand carcinogenesis and to identify novel targets for treatment. As genomic rearrangements require excision of DNA, we hypothesized that an elevated endonuclease activity may induce recombination and subsequent genomic instability in cancer cells. We developed a plasmid degradation assay that confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease the degradation assay was carried out in the presence of specific endonuclease inhibitors, which identified apurinic/apyrimidinic endonuclease (Ape1 and Ape2) as the predominant endonucleases in mediating increased endonuclease activity in MM. Gene expression analysis confirmed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Both immunocytochemistry and western blot analyses confirmed upregulation of Ape1 protein in all MM cell lines and patient samples. Next, we investigated the role of elevated APE endonuclease activity in DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. To investigate the role of elevated AP endonuclease activity in MM, we cultured myeloma cells in the presence of methoxyamine (MX), which specifically inhibits AP endonuclease activity, and evaluated its effect on HR activity and genome-wide appearance of new mutations. Exposure of intact myeloma cells to MX resulted in > 90% inhibition of HR activity and a significant (71±10.9%; p<0.05) reduction in the appearance of new mutations compared to untreated cells, as assessed by genome-wide loss of heterozygosity (LOH) assay (Affymetrix). We also evaluated the effects of overexpression of Ape1 & 2 in normal fibroblasts which have low endonuclease activity. The transgenic upregulation of AP endonucleases (Ape1 and Ape2) in normal cells led to a significant increase in the lecombination activity, leading to a marked mutational instability as indicated by the appearance of over 20,063 and 20,143 new LOH loci per 100,000 polymorphic regions examined throughout the genome, at population doublings 25 and 50 respectively. Mutational instability was also associated with chromosomal instability confirmed by spectral karyotyping of these cells showing significant numerical and structural chromosomal abnormalities. These changes were associated with indefinite growth of cells and formation of tumors when injected in SCID mice. These data suggest that elevated AP endonuclease may be responsible for mutational and chromosomal instabilities, leading to progression of myeloma.

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Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Blood ◽

10.1182/blood.v108.11.5080.5080 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5080-5080

Author(s):

Shankaranarayana Paneesha ◽

Raghu Adya ◽

Hemali Khanji ◽

Ed Leung ◽

C. Vijayasekar ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Endothelial Cells ◽

Mrna Expression ◽

Cell Lines ◽

Myeloma Cell ◽

Myeloma Cells ◽

Human Myeloma Cell

Abstract Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection. Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE). Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

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Stroma cells and Monocytes make Multiple Myeloma Cells Active In Bone Marrow Microenviroment

Blood ◽

10.1182/blood.v122.21.5333.5333 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5333-5333

Author(s):

Hiroshi Ikeda ◽

Tadao Ishida ◽

Toshiaki Hayashi ◽

Yuka Aoki ◽

Yasuhisa Shinomura

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Cell Contact ◽

Drug Induced ◽

Myeloma Cells

Abstract The Bone marrow (BM) microenvironment plays crucial role in pathogenesis of multiple myeloma　(MM). Paracrine secretion of cytokines in BM stromal cells promotes multiple myeloma cell proliferation and protects against drug-induced cytotoxicity. In current study, monocytes, component of BM cells, can directly promote mesenchymal stem cells osteogenic differentiation through cell contact interactions. Down-regulation of inhibitors such as DKK1 drives the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocytes as a potential niche component that supports myeloma cells. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BM stromal cells, monocytes, or a combination of BM stromal cells and monocytes. Consistently, we observed increased proliferation of MM cell lines in the presence of either BM stromal cells or monocytes compared to cell line-only control. Furthermore, the co-culture of BM stromal cells plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Receptor Tyrosine Kinase AXL: A Potential Strategy to Counter Immune Suppression and Dormancy in Multiple Myeloma

Blood ◽

10.1182/blood-2019-126834 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4335-4335

Author(s):

Kim De Veirman ◽

Siyang Yan ◽

Ken Maes ◽

Nathan De Beule ◽

Sylvia Faict ◽

...

Keyword(s):

Multiple Myeloma ◽

Overall Survival ◽

Tyrosine Kinase ◽

Cell Lines ◽

Receptor Tyrosine Kinase ◽

Single Agent ◽

Myeloid Cells ◽

Myeloma Cell ◽

Myeloma Cells

Introduction The AXL receptor tyrosine kinase (AXL) has emerged as a promising therapeutic target for cancer therapy. Recent studies revealed a crucial role of AXL signaling in proliferation, survival, dormancy and therapy resistance in different cancers including lung cancer, hepatocellular cancer and AML. In this study, we aimed to investigate the role of AXL in Multiple Myeloma (MM), focusing on myeloma cell dormancy and AXL expression in different cellular components of the bone marrow microenvironment. Material & Methods To investigate dormancy, we used the syngeneic murine 5TGM1 MM model. 5TGM1-GFP+cells were DiD-labeled and injected intravenously in naïve C57BL/KaLwRij mice. At end-stage, GFP+DiD+('dormant', non-proliferating) and GFP+DiD-('proliferating') MM cells were analyzed by flow cytometry for AXL expression. In addition, AXL expression was also analyzed in CD11b+ myeloid cells and in in vitrogenerated macrophages from the 5TMM model. The effects of AXL inhibition by R428 (BGB324|Bemcentinib, Sigma-Aldrich), a highly potent and AXL-specific small molecular inhibitor, on viability and induced apoptosis of MM cells was determined by Cell Titer Glo and AnnexinV/7AAD staining respectively. AXL expression in human myeloma cell lines (HMCL) (JJN3, U266 and LP-1) and murine 5TGM1 cells was analyzed by qRT-PCR and cytospin stainings. Patient cohorts (TT2/TT3) were used to correlate AXL expression and overall survival. Plasma of healthy donors and MM patients was analyzed by ELISA (R&D). Results Using the in vivo5TGM1 dormancy model, we demonstrated an increased expression of AXL (4x higher) in dormant MM cells compared to proliferating MM cells (n=3, p<0,05). Myeloma cell lines (JJN3, U266, 5TGM1) had a very low AXL expression, however, treatment with melphalan induced a more than twofold increase in AXL expression (n=3, p<0.05). The combination of melphalan and R428 significantly increased apoptosis of JJN3 (>10%), U266 (>20%) and LP-1 (>10%) cells compared to single agent therapy (n=6) (p<0.01). Using patient cohorts, we observed that AXL expression correlated with a good overall survival (p=0.006). In addition, plasma samples of patients (n=31) showed a decreased expression of AXL compared to samples of healthy controls (n=9) (p<0.001). This confirms our hypothesis that AXL is associated with dormancy and therefore correlates with a better overall survival. In a second part, we investigated AXL expression in 5TMM-derived myeloid cells and macrophages (n=3). We observed a high expression of AXL in myeloid derived suppressor cells and tumor associated macrophages compared to myeloma cells. In addition, we observed that myeloid cells were much more sensitive to R428 compared to MM cells (n=5, p>0.01). Conclusion We observed that AXL is highly expressed in dormant MM cells and environmental myeloid cells. Despite its association with a good prognosis in MM, AXL serves as an interesting target to eradicate dormant myeloma cells as AXL inhibitors affect viability and induce apoptosis of myeloma cells, especially in combination with melphalan. Therefore, AXL can be considered as a new therapeutic strategy, to target both the immunosuppressive myeloid cells and the residual cancer cells in MM patients. Disclosures No relevant conflicts of interest to declare.

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Role Of RECQ1 Helicase In Multiple Myeloma Biology and Drug Resistance

Blood ◽

10.1182/blood.v122.21.1889.1889 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1889-1889

Author(s):

Elena Viziteu ◽

Bernard Klein ◽

Angelique Bruyer ◽

Dirk Hose ◽

Hartmut Goldschmidt ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Lines ◽

Cellular Proliferation ◽

Alkylating Agents ◽

Myeloma Cell ◽

Mitotic Catastrophe ◽

Myeloma Cells ◽

Dnmt Inhibitor ◽

Promising Therapeutic Approach

Abstract Multiple Myeloma (MM) is a still lethal disease in 2013 characterized by the accumulation in the bone marrow of a clone of malignant plasma cells. Recent studies have shown that epigenetic modifications play a role by silencing various cancer-related genes in MM. We initiated a microarray-based genome-wide screen for genes responding to DNMT inhibition in MM cells and built a “DNA methylation gene score” that makes it possible identification of myeloma patients that will be sensitive to DNMT inhibitors. Among the genes regulated by DNMT inhibitor and associated with the worst prognostic value in patients, RECQ1 was identified. RECQ helicase are DNA unwinding enzymes involved in the maintenance of chromosome stability. RECQ1 is highly expressed in various types of solid tumors. RECQ1 silencing in cancer cells results in mitotic catastrophe and prevents tumor growth in murine models. In glioblastoma cells, depletion of RECQ1 induces reduction in cellular proliferation, spontaneous γ-H2AX foci formation and hypersensitivity to drugs. Furthermore, it was described that RECQ1 protein could interact with MSH proteins, RAD51 and PARP1 involved in DNA repair pathways. RECQ1 protein is expressed in human myeloma cell lines (HMCLs) and primary myeloma cells of patients. In four HMCLs (XG2, XG7, XG19 and LP1), RECQ1 was downregulated by conditional shRNA expression through lentiviral delivery. RECQ1 knock down inhibits growth of myeloma cells, induces 53BP1 foci formation and apoptosis. RECQ1 depletion sensitizes myeloma cells to DNA alkylating agent (melphalan) but not to corticosteroid (dexamethasone) or proteasome inhibitor (bortezomib). Using immunoprecipitation of myeloma cell nuclear proteins with anti-RECQ1 antibody, RECQ1 was shown to interact with PARP1 but not RAD51 or MSH2. An increased association of the two proteins was found upon DNA damages induced by melphalan. In agreement, RECQ1 depletion sensitizes myeloma cell lines to the PJ34 hydrochloride hydrate PARP inhibitor. In conclusion, RECQ1 could represent a biomarker of drug resistance in MM, which is targeted by DNMT inhibitor. This suggests association of alkylating agents and/or PARP inhibitors with DNMT inhibitor may represent a promising therapeutic approach. Disclosures: Goldschmidt: Celgene and Janssen: Membership on an entity’s Board of Directors or advisory committees.

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N-Cadherin Expressing MM Cells Form Calcium-Dependent, Adherent Junctions That Likely Contribute to Focal Lesions and Nodular Growth of Multiple Myeloma Cells

Blood ◽

10.1182/blood.v116.21.979.979 ◽

2010 ◽

Vol 116 (21) ◽

pp. 979-979

Author(s):

Ya-Wei Qiang ◽

Bo Hu ◽

Yu Chen ◽

Erming Tian ◽

Joshua Epstein ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Bone Destruction ◽

Dependent Manner ◽

Focal Lesions ◽

Myeloma Cells ◽

Calcium Dependent ◽

Nodular Growth ◽

E Cadherin

Abstract Abstract 979 E-cadherin-mediated adhesion regulates homeostasis in tissues of epithelial origin and homotypic N-cadherin interactions are central to the interaction of hematopoietic stem cells and the endosteal niche. Loss of E-cadherin in carcinomas is characteristic of the epithelial-to-mesenchymal transition and metastasis. The loss of E-cadherin, causes the release of b-catenin from the adherent complex, increased nuclear translocation, and increased transcriptional activity of b-catenin/TCF. We and others have demonstrated that alterations in the Wnt/b-catenin pathway exists in multiple myeloma, a malignancy of terminally differentiated antibody secreting plasma cells. The first evidence of this deregulation came from studies showing that MM cells secrete the potent Wnt/b-catenin signaling inhibitor DKK1. While it is now clear that DKK1 mediated suppression of Wnt/b-catenin in the bone marrow contributes to the decoupling of bone formation, the role of Wnt/b-catenin in normal plasma cell development and myelomagenesis is less clear and often controversial. Myeloma cells grow exclusively in the bone marrow. This growth is characterized as being interstitial or nodular, with most disease exhibiting a mixed pattern. Nodular growth, recognized as focal lesions (FL) on MRI, characterizes the conversion of MGUS to symptomatic MM. Consistently, DKK1 levels are highest in CD138 cells isolated from FL where nodular tumor growth and bone destruction may be linked by Wnt/b-catenin suppression in MM cells and local microenvironment. Given the central role of classical cadherins in promoting cell-cell adhesion and regulating b-catenin, we hypothesized that abnormal expression of cadherins might play a direct role in myelomagenesis. Cell lysates were prepared from 24 MM cell lines and from CD138+ cells from the BM of eight patients with MM. Immunoblotting was performed with antibodies specific to human N-cadherin protein. N-cadherin protein was observed in more than 82% of MM cell lines, with high protein levels in 55% of the cell lines. Similar levels of N-cadherin protein were seen in primary myeloma cells from the eight MM patients: two patients showed the highest levels of N-cadherin protein, two showed intermediate levels, and three weak levels; N-cadherin protein was absent in one patient. Similar results were obtained with three color-flow cytometry analysis of primary MM BM. To determine whether N-cadherin mediated MM cell interactions, a cell aggregation assay using GFP-expressing, N-cadherin+ MM cells observed under fluorescence microscopy was employed. When N-cadherin+ JJN3 cells were cultured in normal growth medium, they aggregated to form clusters. Similar results were observed in other MM cell lines, including OPM-2 and KMS-28-BM. Addition of a neutralizing N-cadherin antibody to these cultures significantly attenuated aggregation of JJN3 cells compared to control cells in normal medium or cells treated with control IgG. Homotypic N-cadherin interaction forms adherent junctions in a calcium dependent manner. To see if aggregation of MM cells results in the formation of adherent junctions, immunochemical staining was preformed to visualize the N-cadherin protein in OPM-2 cells. N-cadherin protein was clearly observed between myeloma cells in aggregation clusters that were significantly diminished in calcium-free medium. Taken together, these data suggest that N-cadherin induces homotypic adhesion of myeloma cells in a calcium-dependent manner and suggests that calcium release during bone resorption may enhance adherent junctions in MM that may in turn enhance plasma membrane localization of b-catenin. Studies are currently underway to determine whether DKK1 and N-cadherin adherent junctions cooperate to suppress b-catenin nuclear activity in MM cells and all converge to induce the nodular growth pattern and bone destruction often seen in N-cadherin-positive MM. Disclosures: No relevant conflicts of interest to declare.

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Smenamide A Analogues. Synthesis and Biological Activity on Multiple Myeloma Cells

10.20944/preprints201805.0443.v1 ◽

2018 ◽

Author(s):

Alessia Caso ◽

Ilaria Laurenzana ◽

Daniela Lamorte ◽

Stefania Trino ◽

Germana Esposito ◽

...

Keyword(s):

Lung Cancer ◽

Multiple Myeloma ◽

Biological Activity ◽

Cell Lines ◽

Antiproliferative Activity ◽

Structural Features ◽

Synthetic Route ◽

Myeloma Cells ◽

Apoptotic Mechanism

Smenamides are an intriguing class of peptide/polyketide molecules of marine origin showing antiproliferative activity against lung cancer Calu-1 cells at nanomolar concentrations through a clear pro-apoptotic mechanism. To probe the role of the activity-determining structural features, the 16-epi-analogue of smenamide A and eight simplified analogues in the 16-epi series were prepared using a flexible synthetic route. The synthetic analogues were tested on multiple myeloma (MM) cell lines showing that the configuration at C-16 slightly affects the activity, since the 16-epi-derivative is still active at nanomolar concentrations. Interestingly, it was found that the truncated compound 8, mainly composed of the pyrrolinone terminus, was not active while compound 17, essentially lacking the pyrrolinone moiety, was 1000-fold less active than the intact substance and was the most active among all the synthesized compounds.

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IL6 sensitizes prostate cancer to the antiproliferative effect of IFNα2 through IRF9

Endocrine Related Cancer ◽

10.1530/erc-13-0222 ◽

2013 ◽

Vol 20 (5) ◽

pp. 677-689 ◽

Cited By ~ 11

Author(s):

Holger H H Erb ◽

Regina V Langlechner ◽

Patrizia L Moser ◽

Florian Handle ◽

Tineke Casneuf ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Lines ◽

Cellular Proliferation ◽

Tissue Expression ◽

Type I ◽

Regulatory Factor ◽

Quantitative Real Time Pcr ◽

Antiproliferative Effects ◽

Protein Levels

Development and progression of prostate cancer (PCa) are associated with chronic inflammation. The cytokine interleukin 6 (IL6) can influence progression, differentiation, survival, and angiogenesis of PCa. To identify novel pathways that are triggered by IL6, we performed a gene expression profiling of two PCa cell lines, LNCaP and MDA PCa 2b, treated with 5 ng/ml IL6. Interferon (IFN) regulatory factor 9 (IRF9) was identified as one of the most prevalent IL6-regulated genes in both cell lines. IRF9 is a mediator of type I IFN signaling and acts together with STAT1 and 2 to activate transcription of IFN-responsive genes. The IL6 regulation of IRF9 was confirmed at mRNA and protein levels by quantitative real-time PCR and western blot respectively in both cell lines and could be blocked by the anti-IL6 antibody Siltuximab. Three PCa cell lines, PC3, Du-145, and LNCaP-IL6+, with an autocrine IL6 loop displayed high expression of IRF9. A tissue microarray with 36 PCa tissues showed that IRF9 protein expression is moderately elevated in malignant areas and positively correlates with the tissue expression of IL6. Downregulation and overexpression of IRF9 provided evidence for an IFN-independent role of IRF9 in cellular proliferation of different PCa cell lines. Furthermore, expression of IRF9 was essential to mediate the antiproliferative effects of IFNα2. We concluded that IL6 is an inducer of IRF9 expression in PCa and a sensitizer for the antiproliferative effects of IFNα2.

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Promoter Methylation of RASSF1A Associates to Adult Secondary Glioblastomas and Pediatric Glioblastomas

ISRN Neurology ◽

10.5402/2012/576578 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 7

Author(s):

Jorge Muñoz ◽

María del Mar Inda ◽

Paula Lázcoz ◽

Idoya Zazpe ◽

Xing Fan ◽

...

Keyword(s):

Tumor Suppressor ◽

Cell Lines ◽

Tumor Suppressor Genes ◽

Promoter Hypermethylation ◽

Suppressor Genes ◽

Primary Tumors ◽

Specific Pcr ◽

Inactivation Mechanism ◽

Altered Gene ◽

Homozygous Deletions

While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14ARF, and p16INK4A), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14ARF and p16INK4A did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14ARF and p16INK4A, in which other alterations (mutations, homozygous deletions) are prevalent.

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Promoter Methylation ofSFRP3Is Frequent in Hepatocellular Carcinoma

Disease Markers ◽

10.1155/2014/351863 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 9

Author(s):

Ya-Wen Lin ◽

Yu-Lueng Shih ◽

Gi-Shih Lien ◽

Fat-Moon Suk ◽

Chung-Bao Hsieh ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Chronic Hepatitis ◽

Mrna Expression ◽

Cell Lines ◽

Promoter Methylation ◽

Promoter Hypermethylation ◽

Polymerase Chain ◽

Related Proteins ◽

And Control

Oncogenic activation of the Wnt/β-catenin signaling pathway is common in human cancers. The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and have important implications in carcinogenesis. Because there have been no reports about the role ofSFRP3in hepatocellular carcinoma (HCC), we investigated the level of methylation and transcription ofSFRP3. Four HCC cell lines, 60 HCCs, 23 cirrhosis livers, 37 chronic hepatitis livers, and 30 control livers were prescreened forSFRP3promoter methylation by methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing.SFRP3promoter methylation was observed in 100%, 60%, 39.1%, 16.2%, and 0% in HCC cell lines, primary HCCs, cirrhosis livers, chronic hepatitis livers, and control livers, respectively. Demethylation treatment with 5-aza-2′-deoxycytidine in HCC cells restored or increased theSFRP3mRNA expression. We next used quantitative MS-PCR (QMSP) to analyze the methylation level ofSFRP3in 60 HCCs and their corresponding nontumor tissues. Methylation ofSFRP3promoter region in HCCs increased significantly compared with control tissues. There is a positive correlation between promoter hypermethylation andSFRP3mRNA downregulation. Our data suggest that promoter hypermethylation ofSFRP3is a common event in HCCs and plays an important role in regulation ofSFRP3mRNA expression.

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