Gene Summary

Gene:CST6; cystatin E/M
Summary:The cystatin superfamily encompasses proteins that contain multiple cystatin-like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never acquired this inhibitory activity. There are three inhibitory families in the superfamily, including the type 1 cystatins (stefins), type 2 cystatins and the kininogens. The type 2 cystatin proteins are a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions, where they appear to provide protective functions. This gene encodes a cystatin from the type 2 family, which is down-regulated in metastatic breast tumor cells as compared to primary tumor cells. Loss of expression is likely associated with the progression of a primary tumor to a metastatic phenotype. [provided by RefSeq, Jul 2008]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Source:NCBIAccessed: 09 March, 2017


What does this gene/protein do?
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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

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Survival Rate
  • Uterine Cancer
  • Immunohistochemistry
  • Neoplasm Invasiveness
  • Neoplasm Metastasis
  • Receptors, Progesterone
  • Breast Cancer
  • Oligonucleotide Array Sequence Analysis
  • Epigenetics
  • Up-Regulation
  • Receptor, erbB-2
  • Lymphatic Metastasis
  • Cell Proliferation
  • Gene Silencing
  • Sensitivity and Specificity
  • Neoplasm Recurrence, Local
  • Azacitidine
  • Promoter Regions
  • CpG Islands
  • Signal Transduction
  • Base Sequence
  • Cancer DNA
  • Molecular Sequence Data
  • Messenger RNA
  • Biomarkers, Tumor
  • Cluster Analysis
  • Gene Expression Profiling
  • Chromosome 11
  • Cystatins
  • Transfection
  • Breast
  • Staging
  • Tumor Suppressor Proteins
  • Cystatin M
  • Cervical Cancer
  • Cancer Gene Expression Regulation
  • Polymerase Chain Reaction
  • DNA Methylation
  • Tumor Suppressor Gene
Tag cloud generated 09 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (3)

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: CST6 (cancer-related)

Parisi C, Mastoraki S, Markou A, et al.
Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy analysis.
Clin Chim Acta. 2016; 461:156-64 [PubMed] Related Publications
BACKGROUND: Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs.
METHODS: We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors.
RESULTS: In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively.
CONCLUSIONS: Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer.

Li Z, Guo X, Wu Y, et al.
Methylation profiling of 48 candidate genes in tumor and matched normal tissues from breast cancer patients.
Breast Cancer Res Treat. 2015; 149(3):767-79 [PubMed] Related Publications
Gene-specific methylation alterations in breast cancer have been suggested to occur early in tumorigenesis and have the potential to be used for early detection and prevention. The continuous increase in worldwide breast cancer incidences emphasizes the urgent need for identification of methylation biomarkers for early cancer detection and patient stratification. Using microfluidic PCR-based target enrichment and next-generation bisulfite sequencing technology, we analyzed methylation status of 48 candidate genes in paired tumor and normal tissues from 180 Chinese breast cancer patients. Analysis of the sequencing results showed 37 genes differentially methylated between tumor and matched normal tissues. Breast cancer samples with different clinicopathologic characteristics demonstrated distinct profiles of gene methylation. The methylation levels were significantly different between breast cancer subtypes, with basal-like and luminal B tumors having the lowest and the highest methylation levels, respectively. Six genes (ACADL, ADAMTSL1, CAV1, NPY, PTGS2, and RUNX3) showed significant differential methylation among the 4 breast cancer subtypes and also between the ER +/ER- tumors. Using unsupervised hierarchical clustering analysis, we identified a panel of 13 hypermethylated genes as candidate biomarkers that performed a high level of efficiency for cancer prediction. These 13 genes included CST6, DBC1, EGFR, GREM1, GSTP1, IGFBP3, PDGFRB, PPM1E, SFRP1, SFRP2, SOX17, TNFRSF10D, and WRN. Our results provide evidence that well-defined DNA methylation profiles enable breast cancer prediction and patient stratification. The novel gene panel might be a valuable biomarker for early detection of breast cancer.

D'Costa ZC, Higgins C, Ong CW, et al.
TBX2 represses CST6 resulting in uncontrolled legumain activity to sustain breast cancer proliferation: a novel cancer-selective target pathway with therapeutic opportunities.
Oncotarget. 2014; 5(6):1609-20 [PubMed] Free Access to Full Article Related Publications
TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.

Peters I, Dubrowinskaja N, Abbas M, et al.
DNA methylation biomarkers predict progression-free and overall survival of metastatic renal cell cancer (mRCC) treated with antiangiogenic therapies.
PLoS One. 2014; 9(3):e91440 [PubMed] Free Access to Full Article Related Publications
VEGF-targeted therapy increases both the progression-free (PFS) and overall survival (OS) of patients with metastasized renal cell cancer (mRCC). Identification of molecular phenotypes of RCC could improve risk-stratification and the prediction of the clinical disease course. We investigated whether gene-specific DNA hypermethylation can predict PFS and OS among patients undergoing anti-VEGF-based therapy. Primary tumor tissues from 18 patients receiving targeted therapy were examined retrospectively using quantitative methylation-specific PCR analysis of CST6, LAD1, hsa-miR-124-3, and hsa-miR-9-1 CpG islands. PFS and OS were analyzed for first-line and sequential antiangiogenic therapies using the log rank statistics. Sensitivity and specificity were determined for predicting first-line therapy failure. Hypermethylation of CST6 and LAD1 was associated with both a shortened PFS (log rank p = 0.009 and p = 0.004) and OS (p = 0.011 and p = 0.043). The median PFS observed for the high and low methylation groups of CST6 and LAD1 was 2.0 vs.11.4 months. LAD1 methylation had a specificity of 1.0 (95% CI 0.65-1.0) and a sensitivity of 0.73 (95% CI 0.43-0.90) for the prediction of first-line therapy. CST6 and LAD1 methylation are candidate epigenetic biomarkers showing unprecedented association with PFS and OS as well as specificity for the prediction of the response to therapy. DNA methylation markers should be considered for the prospective evaluation of larger patient cohorts in future studies.

Tessema M, Yingling CM, Liu Y, et al.
Genome-wide unmasking of epigenetically silenced genes in lung adenocarcinoma from smokers and never smokers.
Carcinogenesis. 2014; 35(6):1248-57 [PubMed] Free Access to Full Article Related Publications
Lung cancer in never smokers (NS) shows striking demographic, clinicopathological and molecular distinctions from the disease in smokers (S). Studies on selected genetic and epigenetic alterations in lung cancer identified that the frequency and profile of some abnormalities significantly differ by smoking status. This study compared the transcriptome of lung adenocarcinoma cell lines derived from S (n = 3) and NS (n = 3) each treated with vehicle (control), histone deacetylation inhibitor (trichostatin A) or DNA methylation inhibitor (5-aza-2'-deoxycytidine). Among 122 genes reexpressed following 5-aza-2'-deoxycytidine but not trichostatin A treatment in two or more cell lines (including 32 genes in S-only and 12 NS-only), methylation was validated for 80% (98/122 genes). After methylation analysis of 20 normal tissue samples and 14 additional non-small cell lung cancer cell lines (total 20), 39 genes frequently methylated in normal (>20%, 4/20) and 21 genes rarely methylated in non-small cell lung cancer (≤10%, 2/20) were excluded. The prevalence for methylation of the remaining 38 genes in lung adenocarcinomas from S (n = 97) and NS (n = 75) ranged from 8-89% and significantly differs between S and NS for CPEB1, CST6, EMILIN2, LAYN and MARVELD3 (P < 0.05). Furthermore, methylation of EMILIN2, ROBO3 and IGDCC4 was more prevalent in advanced (Stage II-IV, n = 61) than early (Stage I, n = 110) tumors. Knockdown of MARVELD3, one of the novel epigenetically silenced genes, by small interfering RNA significantly reduced anchorage-independent growth of lung cancer cells (P < 0.001). Collectively, this study has identified multiple, novel, epigenetically silenced genes in lung cancer and provides invaluable resources for the development of diagnostic and prognostic biomarkers.

Sandhu R, Rivenbark AG, Mackler RM, et al.
Dysregulation of microRNA expression drives aberrant DNA hypermethylation in basal-like breast cancer.
Int J Oncol. 2014; 44(2):563-72 [PubMed] Free Access to Full Article Related Publications
Basal-like breast cancers frequently express aberrant DNA hypermethylation associated with concurrent silencing of specific genes secondary to DNMT3b overexpression and DNMT hyperactivity. DNMT3b is known to be post-transcriptionally regulated by microRNAs. The objective of the current study was to determine the role of microRNA dysregulation in the molecular mechanism governing DNMT3b overexpression in primary breast cancers that express aberrant DNA hypermethylation. The expression of microRNAs (miRs) that regulate (miR-29a, miR-29b, miR-29c, miR-148a and miR-148b) or are predicted to regulate DNMT3b (miR‑26a, miR-26b, miR-203 and miR-222) were evaluated among 70 primary breast cancers (36 luminal A-like, 13 luminal B-like, 5 HER2‑enriched, 16 basal-like) and 18 normal mammoplasty tissues. Significantly reduced expression of miR-29c distinguished basal-like breast cancers from other breast cancer molecular subtypes. The expression of aberrant DNA hypermethylation was determined in a subset of 33 breast cancers (6 luminal A-like, 6 luminal B-like, 5 HER2-enriched and 16 basal-like) through examination of methylation‑sensitive biomarker gene expression (CEACAM6, CDH1, CST6, ESR1, GNA11, MUC1, MYB, TFF3 and SCNN1A), 11/33 (33%) cancers exhibited aberrant DNA hypermethylation including 9/16 (56%) basal-like cancers, but only 2/17 (12%) non-basal-like cancers (luminal A-like, n=1; HER2-enriched, n=1). Breast cancers with aberrant DNA hypermethylation express diminished levels of miR-29a, miR-29b, miR-26a, miR-26b, miR-148a and miR-148b compared to cancers lacking aberrant DNA hypermethylation. A total of 7/9 (78%) basal-like breast cancers with aberrant DNA hypermethylation exhibit diminished levels of ≥6 regulatory miRs. The results show that i) reduced expression of miR-29c is characteristic of basal-like breast cancers, ii) miR and methylation-sensitive gene expression patterns identify two subsets of basal-like breast cancers, and iii) the subset of basal-like breast cancers with reduced expression of multiple regulatory miRs express aberrant DNA hypermethylation. Together, these findings strongly suggest that the molecular mechanism governing the DNMT3b-mediated aberrant DNA hypermethylation in primary breast cancer involves the loss of post-transcriptional regulation of DNMT3b by regulatory miRs.

Roll JD, Rivenbark AG, Sandhu R, et al.
Dysregulation of the epigenome in triple-negative breast cancers: basal-like and claudin-low breast cancers express aberrant DNA hypermethylation.
Exp Mol Pathol. 2013; 95(3):276-87 [PubMed] Related Publications
A subset of human breast cancer cell lines exhibits aberrant DNA hypermethylation that is characterized by hyperactivity of the DNA methyltransferase enzymes, overexpression of DNMT3b, and concurrent methylation-dependent silencing of numerous epigenetic biomarker genes. The objective of this study was to determine if this aberrant DNA hypermethylation (i) is found in primary breast cancers, (ii) is associated with specific breast cancer molecular subtypes, and (iii) influences patient outcomes. Analysis of epigenetic biomarker genes (CDH1, CEACAM6, CST6, ESR1, GNA11, MUC1, MYB, SCNN1A, and TFF3) identified a gene expression signature characterized by reduced expression levels or loss of expression among a cohort of primary breast cancers. The breast cancers that express this gene expression signature are enriched for triple-negative subtypes - basal-like and claudin-low breast cancers. Methylation analysis of primary breast cancers showed extensive promoter hypermethylation of epigenetic biomarker genes among triple-negative breast cancers, compared to other breast cancer subclasses where promoter hypermethylation events were less frequent. Furthermore, triple-negative breast cancers either did not express or expressed significantly reduced levels of protein corresponding to methylation-sensitive biomarker gene products. Together, these findings suggest strongly that loss of epigenetic biomarker gene expression is frequently associated with gene promoter hypermethylation events. We propose that aberrant DNA hypermethylation is a common characteristic of triple-negative breast cancers and may represent a fundamental biological property of basal-like and claudin-low breast cancers. Kaplan-Meier analysis of relapse-free survival revealed a survival disadvantage for patients with breast cancers that exhibit aberrant DNA hypermethylation. Identification of this distinguishing trait among triple-negative breast cancers forms the basis for development of new rational therapies that target the epigenome in patients with basal-like and claudin-low breast cancers.

Tzadok S, Caspin Y, Hachmo Y, et al.
Directionality of noncoding human RNAs: how to avoid artifacts.
Anal Biochem. 2013; 439(1):23-9 [PubMed] Related Publications
Inactivation of tumor suppressor and metastasis suppressor genes via epigenetic silencing is a frequent event in human cancers. Recent work has shown new mechanisms of epigenetic silencing, based on the occurrence of long noncoding promoter-spanning antisense and/or sense RNAs (lncRNAs), which constitute part of chromatin silencing complexes. Using reverse transcription polymerase chain reaction (RT-PCR), we have started to scan "triple negative" and Her2-overexpressing breast cancer cell lines for directional/bidirectional transcription through promoters of tumor suppressor and metastasis suppressor genes known to be epigenetically silenced in vivo. Surprisingly, we found that RT-PCR-amplified products were obtained at high frequency in the absence of exogenous primers. These amplified products resulted from RT priming via transcripts originating from promoter or upstream spanning regions. Consequently, this priming overruled directionality determination and led to false detection-identification of such lncRNAs. We show that this prevalent "no primer" artifact can be eliminated by treating the RNA preparations with periodate, performing RT reactions at highly elevated temperatures, or a combination of both. These experimental improvements enabled determination of the presence and directionality of individual promoter-spanning long noncoding RNAs with certainty. Examples for the BRMS1 metastasis suppressor gene, as well as RAR-β2 and CST6 human tumor suppressor genes, in breast carcinoma cell lines are presented.

Dimitrakopoulos L, Vorkas PA, Georgoulias V, Lianidou ES
A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples.
BMC Cancer. 2012; 12:486 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.
METHODS: We designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.
RESULTS: The developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.
CONCLUSIONS: The developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.

Chimonidou M, Tzitzira A, Strati A, et al.
CST6 promoter methylation in circulating cell-free DNA of breast cancer patients.
Clin Biochem. 2013; 46(3):235-40 [PubMed] Related Publications
OBJECTIVES: We have recently shown that detection of CST6 promoter methylation in primary breast tumors can provide important prognostic information in patients with operable breast cancer and that CST6 promoter is also methylated in Circulating Tumor Cells (CTC). In this study we evaluated the presence of CST6 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of breast cancer patients.
DESIGN AND METHODS: Our study material consisted of: a) a pilot testing group of 27 patients with stage I-III operable breast cancer, 46 patients with verified metastasis and 37 healthy donors and b) an independent cohort of 123 consecutive stage I-III operable breast cancer patients. Methylated and unmethylated CST6 promoter sequences were detected by using methylation-specific PCR (MSP). CST6 immunohistochemical detection was performed in 20 corresponding primary tumor tissues.
RESULTS: In the pilot testing group, CST6 promoter was methylated in 8/27 (29.6%) operable breast cancer patients, in 6/46 (13.0%) patients with verified metastasis but none of 37 healthy individuals (0%). In the independent cohort, 49/123 (39.8%) operable breast cancer patients were found positive. During the follow up period, 25/123 (20.3%) patients relapsed and 9/123 (7.3%) died. CST6 was methylated in cfDNA of 13/25 (52%) patients that relapsed and in 3/9 (33.3%) patients that died.
CONCLUSIONS: CST6 promoter is highly methylated in cfDNA of breast cancer patients, but not in healthy individuals. CST6 promoter methylation in cfDNA, should be prospectively validated as a novel plasma tumor biomarker for breast cancer in a large cohort of breast cancer patients.

Huang J, Wang L, Jiang M, et al.
PTHLH coupling upstream negative regulation of fatty acid biosynthesis and Wnt receptor signal to downstream peptidase activity-induced apoptosis network in human hepatocellular carcinoma by systems-theoretical analysis.
J Recept Signal Transduct Res. 2012; 32(5):250-6 [PubMed] Related Publications
Studies were done on the analysis of biological processes in the same high expression (fold change ≥ 2) PTHLH-activated feedback negative regulation-mediated apoptosis gene ontology (GO) network of human hepatocellular carcinoma (HCC) compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues [hepatitis B virus (HBV) or hepatitis C virus (HCV) infection]. We proposed PTHLH-activated network that upstream included the regulation of apoptosis, signal transduction resulting in induction of apoptosis, signal transduction by p53 class mediator resulting in transcription of p21 class mediator, negative regulation of centriole replication, negative regulation of fatty acid biosynthesis, negative regulation of Wnt receptor signaling pathway, anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, apoptosis, induction of apoptosis, and negative regulation of phosphorylation. Downstream-network negative regulation of peptidase activity, anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, apoptosis, induction of apoptosis and negative regulation of phosphorylation, as a result of coupling upstream negative regulation of fatty acid biosynthesis and Wnt receptor signal to downstream peptidase activity-induced apoptosis in HCC. Our hypothesis was verified by the different PTHLH-activated feedback negative regulation-mediated apoptosis GO network of HCC compared with the corresponding inhibited GO network of no-tumor hepatitis/cirrhotic tissues, or the same compared with the corresponding inhibited GO network of HCC. PTHLH coupling upstream negative regulation of fatty acid biosynthesis and Wnt receptor signal to downstream peptidase activity-induced apoptosis network was constructed that upstream BRCA1, DKK1, BUB1B activated PTHLH, and downstream PTHLH-activated CST6, BUB1B, NTN1, PHLDA2 in HCC from GEO data set using gene regulatory network inference method and our programing.

Barekati Z, Radpour R, Lu Q, et al.
Methylation signature of lymph node metastases in breast cancer patients.
BMC Cancer. 2012; 12:244 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Invasion and metastasis are two important hallmarks of malignant tumors caused by complex genetic and epigenetic alterations. The present study investigated the contribution of aberrant methylation profiles of cancer related genes, APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14 (ARF), P16 (CDKN2A), P21 (CDKN1A), PTEN, and TIMP3, in the matched axillary lymph node metastasis in comparison to the primary tumor tissue and the adjacent normal tissue from the same breast cancer patients to identify the potential of candidate genes methylation as metastatic markers.
METHODS: The quantitative methylation analysis was performed using the SEQUENOM's EpiTYPER™ assay which relies on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
RESULTS: The quantitative DNA methylation analysis of the candidate genes showed higher methylation proportion in the primary tumor tissue than that of the matched normal tissue and the differences were significant for the APC, BIN1, BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 promoter regions (P<0.05). Among those candidate methylated genes, APC, BMP6, BRCA1 and P16 displayed higher methylation proportion in the matched lymph node metastasis than that found in the normal tissue (P<0.05). The pathway analysis revealed that BMP6, BRCA1 and P16 have a role in prevention of neoplasm metastasis.
CONCLUSIONS: The results of the present study showed methylation heterogeneity between primary tumors and metastatic lesion. The contribution of aberrant methylation alterations of BMP6, BRCA1 and P16 genes in lymph node metastasis might provide a further clue to establish useful biomarkers for screening metastasis.

Jin L, Zhang Y, Li H, et al.
Differential secretome analysis reveals CST6 as a suppressor of breast cancer bone metastasis.
Cell Res. 2012; 22(9):1356-73 [PubMed] Free Access to Full Article Related Publications
Bone metastasis is a frequent complication of breast cancer and a common cause of morbidity and mortality from the disease. During metastasis secreted proteins play crucial roles in the interactions between cancer cells and host stroma. To characterize the secreted proteins that are associated with breast cancer bone metastasis, we preformed a label-free proteomic analysis to compare the secretomes of four MDA-MB-231 (MDA231) derivative cell lines with varied capacities of bone metastasis. A total of 128 proteins were found to be consistently up-/down-regulated in the conditioned medium of bone-tropic cancer cells. The enriched molecular functions of the altered proteins included receptor binding and peptidase inhibition. Through additional transcriptomic analyses of breast cancer cells, we selected cystatin E/M (CST6), a cysteine protease inhibitor down-regulated in bone-metastatic cells, for further functional studies. Our results showed that CST6 suppressed the proliferation, colony formation, migration and invasion of breast cancer cells. The suppressive function against cancer cell motility was carried out by cancer cell-derived soluble CST6. More importantly, ectopic expression of CST6 in cancer cells rescued mice from overt osteolytic metastasis and deaths in the animal study, while CST6 knockdown markedly enhanced cancer cell bone metastasis and shortened animal survival. Overall, our study provided a systemic secretome analysis of breast cancer bone tropism and established secreted CST6 as a bona fide suppressor of breast cancer osteolytic metastasis.

Chimonidou M, Strati A, Tzitzira A, et al.
DNA methylation of tumor suppressor and metastasis suppressor genes in circulating tumor cells.
Clin Chem. 2011; 57(8):1169-77 [PubMed] Related Publications
BACKGROUND: Circulating tumor cells (CTCs) are associated with prognosis in a variety of human cancers and have been proposed as a liquid biopsy for follow-up examinations. We show that tumor suppressor and metastasis suppressor genes are epigenetically silenced in CTCs isolated from peripheral blood of breast cancer patients.
METHODS: We obtained peripheral blood from 56 patients with operable breast cancer, 27 patients with verified metastasis, and 23 healthy individuals. We tested DNA extracted from the EpCAM-positive immunomagnetically selected CTC fraction for the presence of methylated and unmethylated CST6, BRMS1, and SOX17 promoter sequences by methylation-specific PCR (MSP). All samples were checked for KRT19 (keratin 19, formerly CK-19) expression by reverse-transcription quantitative PCR.
RESULTS: In CTCs of patients with operable breast cancer, promoter methylation of CST6 was observed in 17.9%, BRMS1 in 32.1%, and SOX17 in 53.6% of patients. In CTCs of patients with verified metastasis, promoter methylation of CST6 was observed in 37.0%, BRMS1 in 44.4%, and SOX17 in 74.1%. In healthy individuals, promoter methylation of CST6 was observed in 4.3%, BRMS1 in 8.7%, and SOX17 in 4.3%. DNA methylation of these genes for both operable and metastatic breast cancer was significantly different from that of the control population.
CONCLUSIONS: DNA methylation of tumor suppressor and metastasis suppressor genes is a hallmark of CTCs and confirms their heterogeneity. Our findings add a new dimension to the molecular characterization of CTCs and may underlie the acquisition of malignant properties, including their stem-like phenotype.

Radpour R, Barekati Z, Kohler C, et al.
Hypermethylation of tumor suppressor genes involved in critical regulatory pathways for developing a blood-based test in breast cancer.
PLoS One. 2011; 6(1):e16080 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Aberrant DNA methylation patterns might be used as a biomarker for diagnosis and management of cancer patients.
METHODS AND FINDINGS: To achieve a gene panel for developing a breast cancer blood-based test we quantitatively assessed the DNA methylation proportion of 248 CpG sites per sample (total of 31,248 sites in all analyzed samples) on 10 candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P16, P21 and TIMP3). The number of 126 samples consisting of two different cohorts was used (first cohort: plasma samples from breast cancer patients and normal controls; second cohort: triple matched samples including cancerous tissue, matched normal tissue and serum samples). In the first cohort, circulating cell free methylated DNA of the 8 tumor suppressor genes (TSGs) was significantly higher in patients with breast cancer compared to normal controls (P<0.01). In the second cohort containing triple matched samples, seven genes showed concordant hypermethylated profile in tumor tissue and serum samples compared to normal tissue (P<0.05). Using eight genes as a panel to develop a blood-based test for breast cancer, a sensitivity and specificity of more than 90% could be achieved in distinguishing between tumor and normal samples.
CONCLUSIONS: Our study suggests that the selected TSG panel combined with the high-throughput technology might be a useful tool to develop epigenetic based predictive and prognostic biomarker for breast cancer relying on pathologic methylation changes in tumor tissue, as well as in circulation.

Ko E, Park SE, Cho EY, et al.
Cystatin M loss is associated with the losses of estrogen receptor, progesterone receptor, and HER4 in invasive breast cancer.
Breast Cancer Res. 2010; 12(6):R100 [PubMed] Free Access to Full Article Related Publications
INTRODUCTION: This study was aimed at understanding the clinicopathological significance of cystatin M loss, and investigating possible factors responsible for cystatin M loss in breast cancer.
METHODS: The expression of estrogen receptor (ER), progesterone receptor (PR), HER2, HER4, and cystatin M was retrospectively analyzed using immunohistochemistry in 117 patients with ductal carcinoma in situ (DCIS) and in 175 patients with invasive breast cancer (IBC). The methylation status of CST6 gene encoding cystatin M was evaluated using methylation-specific polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissues from 292 participants and using pyrosequencing in fresh-frozen tumor and matched normal tissues from 51 IBC patients.
RESULTS: Cystatin M loss was found in 9 (8%) of 117 patients with DCIS and in 99 (57%) of 175 with invasive breast cancer (IBC) (P < 0.0001). Cystatin M loss was found in 58 (57%) of 101 HER2-negative IBCs and in 41 (55%) of 74 HER2-positive IBCs, and this difference was not statistically significant (P = 0.97). However, cystatin M loss was significantly associated with the loss of ER (P = 0.01), PR (P = 0.002), and HER4 (P = 0.003) in IBCs. Cystatin M loss occurred in 34 (76%) of the 45 HER4-negative IBCs and in 65 (50%) of the 130 HER4-positive IBCs. Multivariate analysis showed that cystatin M loss occurred at a 3.57 times (95% CI = 1.28 to 9.98; P = 0.01) higher prevalence in the triple-negative IBCs of ER, PR, and HER4 than in other subtypes, after adjusting for age. The quantity of CST6 methylation was associated with ER loss (P = 0.0002) in IBCs but not with the loss of PR (P = 0.64) or HER4 (P = 0.87).
CONCLUSIONS: The present study suggests that cystatin M loss may be associated with the losses of ER, PR, and HER4 in IBC.

Morris MR, Ricketts C, Gentle D, et al.
Identification of candidate tumour suppressor genes frequently methylated in renal cell carcinoma.
Oncogene. 2010; 29(14):2104-17 [PubMed] Free Access to Full Article Related Publications
Promoter region hyermethylation and transcriptional silencing is a frequent cause of tumour suppressor gene (TSG) inactivation in many types of human cancers. Functional epigenetic studies, in which gene expression is induced by treatment with demethylating agents, may identify novel genes with tumour-specific methylation. We used high-density gene expression microarrays in a functional epigenetic study of 11 renal cell carcinoma (RCC) cell lines. Twenty-eight genes were then selected for analysis of promoter methylation status in cell lines and primary RCC. Eight genes (BNC1, PDLIM4, RPRM, CST6, SFRP1, GREM1, COL14A1 and COL15A1) showed frequent (>30% of RCC tested) tumour-specific promoter region methylation. Hypermethylation was associated with transcriptional silencing. Re-expression of BNC1, CST6, RPRM and SFRP1 suppressed the growth of RCC cell lines and RNA interference knock-down of BNC1, SFRP1 and COL14A1 increased the growth of RCC cell lines. Methylation of BNC1 or COL14A1 was associated with a poorer prognosis independent of tumour size, stage or grade. The identification of these epigenetically inactivated candidate RCC TSGs can provide insights into renal tumourigenesis and a basis for developing novel therapies and biomarkers for prognosis and detection.

Kioulafa M, Balkouranidou I, Sotiropoulou G, et al.
Methylation of cystatin M promoter is associated with unfavorable prognosis in operable breast cancer.
Int J Cancer. 2009; 125(12):2887-92 [PubMed] Related Publications
The methylation status of cystatin M (CST6) gene in breast tumors was investigated and its prognostic significance as a novel breast cancer biomarker was evaluated. Using methylation-specific PCR (MSP), CST6 promoter methylation was examined in 134 formalin fixed paraffin-embedded tissues (FFPEs): 10 pairs of breast tumors and their surrounding normal tissues, 10 breast fibroadenomas, 11 normal breast tissues and 93 breast tumors. Methylation of CST6 promoter was observed in 2/21 (9.5%) noncancerous breast tissues, 1/10 (10%) benign breast tumors (fibroadenomas) and 52 (55.9%) operable breast cancer tumor samples. CST6 was rarely methylated in the normal tissue surrounding the tumor (10%). During the follow-up period, 24 (25.8%) patients relapsed and 19 (20.4%) died. CST6 methylation was detected in 19 (79.2%) of patients who relapsed and in 15 (78.9%) of patients who died. Disease-free-interval (DFI) and overall survival (OS) were significantly associated with CST6 promoter methylation (p=0.004 and p=0.001 respectively). Multivariate analysis revealed that CST6 methylation is an independent prognostic factor for DFI (HR=3.484; 95% CI: 1.155-10.511; p=0.027). and OS (HR=9.190; 95% CI: 1.989-42.454; p=0.004). CST6 promoter methylation status in tumor cells seems to provide important prognostic information in operable breast cancer and merits to be further evaluated and validated in a larger cohort of patients.

Radpour R, Kohler C, Haghighi MM, et al.
Methylation profiles of 22 candidate genes in breast cancer using high-throughput MALDI-TOF mass array.
Oncogene. 2009; 28(33):2969-78 [PubMed] Related Publications
Alterations of DNA methylation patterns have been suggested as biomarkers for diagnostics and therapy of cancers. Every novel discovery in the epigenetic landscape and every development of an improved approach for accurate analysis of the events may offer new opportunity for the management of patients. Using a novel high-throughput mass spectrometry on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) silico-chips, we determined semiquantitative methylation changes of 22 candidate genes in breast cancer tissues. For the first time we analysed the methylation status of a total of 42 528 CpG dinucleotides on 22 genes in 96 different paraffin-embedded tissues (48 breast cancerous tissues and 48 paired normal tissues). A two-way hierarchical cluster analysis was used to classify methylation profiles. In this study, 10 hypermethylated genes (APC, BIN1, BMP6, BRCA1, CST6, ESRb, GSTP1, P16, P21 and TIMP3) were identified to distinguish between cancerous and normal tissues according to the extent of methylation. Individual assessment of the methylation status for each CpG dinucleotide indicated that cytosine hypermethylation in the cancerous tissue samples was mostly located near the consensus sequences of the transcription factor binding sites. These hypermethylated genes may serve as biomarkers for clinical molecular diagnosis and targeted treatments of patients with breast cancer.

Pulukuri SM, Gorantla B, Knost JA, Rao JS
Frequent loss of cystatin E/M expression implicated in the progression of prostate cancer.
Oncogene. 2009; 28(31):2829-38 [PubMed] Free Access to Full Article Related Publications
Cystatin E/M (CST6) is a natural inhibitor of lysosomal cysteine proteases. Recent studies have shown that experimental manipulation of CST6 expression alters the metastatic behavior of human breast cancer cells. However, the association of CST6 with prostate cancer invasion and progression remains unclear. Here, we show that CST6 is robustly expressed in normal human prostate epithelium, whereas its expression is downregulated in metastatic prostate cell lines and prostate tumor tissues. Treatment of metastatic prostate cell lines with the histone deacetylase inhibitor trichostatin A resulted in significant induction of CST6 mRNA levels and increased CST6 protein expression, indicating that epigenetic silencing may play a role in the loss of CST6 expression observed in prostate cancer. CST6 overexpression in human prostate cancer cells significantly reduced in vitro cell proliferation and matrigel invasion. Furthermore, the results from a bioluminescence tumor/metastasis model showed that the overexpression of CST6 significantly inhibits tumor growth and the incidence of lung metastasis. These results suggest that the downregulation of the CST6 gene is associated with promoter histone modifications and that this association plays an important role in prostate cancer progression during the invasive and metastatic stages of the disease.

Lin HJ, Zuo T, Lin CH, et al.
Breast cancer-associated fibroblasts confer AKT1-mediated epigenetic silencing of Cystatin M in epithelial cells.
Cancer Res. 2008; 68(24):10257-66 [PubMed] Free Access to Full Article Related Publications
The interplay between histone modifications and promoter hypermethylation provides a causative explanation for epigenetic gene silencing in cancer. Less is known about the upstream initiators that direct this process. Here, we report that the Cystatin M (CST6) tumor suppressor gene is concurrently down-regulated with other loci in breast epithelial cells cocultured with cancer-associated fibroblasts (CAF). Promoter hypermethylation of CST6 is associated with aberrant AKT1 activation in epithelial cells, as well as the disabled INNP4B regulator resulting from the suppression by CAFs. Repressive chromatin, marked by trimethyl-H3K27 and dimethyl-H3K9, and de novo DNA methylation is established at the promoter. The findings suggest that microenvironmental stimuli are triggers in this epigenetic cascade, leading to the long-term silencing of CST6 in breast tumors. Our present findings implicate a causal mechanism defining how tumor stromal fibroblasts support neoplastic progression by manipulating the epigenome of mammary epithelial cells. The result also highlights the importance of direct cell-cell contact between epithelial cells and the surrounding fibroblasts that confer this epigenetic perturbation. Because this two-way interaction is anticipated, the described coculture system can be used to determine the effect of epithelial factors on fibroblasts in future studies.

Hosokawa M, Kashiwaya K, Eguchi H, et al.
Over-expression of cysteine proteinase inhibitor cystatin 6 promotes pancreatic cancer growth.
Cancer Sci. 2008; 99(8):1626-32 [PubMed] Related Publications
Pancreatic ductal adenocarcinoma (PDAC) shows the worst mortality among the common malignancies and development of novel therapies for PDAC through identification of good molecular targets is an urgent issue. Among dozens of over-expressing genes identified through our gene-expression profile analysis of PDAC cells, we here report CST6 (Cystatin 6 or E/M) as a candidate of molecular targets for PDAC treatment. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis confirmed over-expression of CST6 in PDAC cells, but no or limited expression of CST6 was observed in normal pancreas and other vital organs. Knock-down of endogenous CST6 expression by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in maintaining viability of PDAC cells. Concordantly, constitutive expression of CST6 in CST6-null cells promoted their growth in vitro and in vivo. Furthermore, the addition of mature recombinant CST6 in culture medium also promoted cell proliferation in a dose-dependent manner, whereas recombinant CST6 lacking its proteinase-inhibitor domain and its non-glycosylated form did not. Over-expression of CST6 inhibited the intracellular activity of cathepsin B, which is one of the putative substrates of CST6 proteinase inhibitor and can intracellularly function as a pro-apoptotic factor. These findings imply that CST6 is likely to involve in the proliferation and survival of pancreatic cancer probably through its proteinase inhibitory activity, and it is a promising molecular target for development of new therapeutic strategies for PDAC.

Oler G, Camacho CP, Hojaij FC, et al.
Gene expression profiling of papillary thyroid carcinoma identifies transcripts correlated with BRAF mutational status and lymph node metastasis.
Clin Cancer Res. 2008; 14(15):4735-42 [PubMed] Free Access to Full Article Related Publications
PURPOSE: To identify papillary thyroid carcinoma (PTC)-associated transcripts, we compared the gene expression profiles of three Serial Analysis of Gene Expression libraries generated from thyroid tumors and a normal thyroid tissue.
EXPERIMENTAL DESIGN: Selected transcripts were validated in a panel of 57 thyroid tumors using quantitative PCR (qPCR). An independent set of 71 paraffin-embedded sections was used for validation using immunohistochemical analysis. To determine if PTC-associated gene expression could predict lymph node involvement, a separate cohort of 130 primary PTC (54 metastatic and 76 nonmetastatic) was investigated. The BRAF(V600E) mutational status was compared with qPCR data to identify genes that might be regulated by abnormal BRAF/MEK/extracellular signal-regulated kinase signaling.
RESULTS: We identified and validated new PTC-associated transcripts. Three genes (CST6, CXCL14, and DHRS3) are strongly associated with PTC. Immunohistochemical analysis of CXCL14 confirmed the qPCR data and showed protein expression in PTC epithelial cells. We also observed that CST6, CXCL14, DHRS3, and SPP1 were associated with PTC lymph node metastasis, with CST6, CXCL14, and SPP1 being positively correlated with metastasis and DHRS3 being negatively correlated. Finally, we found a strong correlation between CST6 and CXCL14 expression and BRAF(V600E) mutational status, suggesting that these genes may be induced subsequently to BRAF activation and therefore may be downstream in the BRAF/MEK/extracellular signal-regulated kinase signaling pathway.
CONCLUSION: CST6, CXCL14, DHRS3, and SPP1 may play a role in PTC pathogenesis and progression and are possible molecular targets for PTC therapy.

Rodenhiser DI, Andrews J, Kennette W, et al.
Epigenetic mapping and functional analysis in a breast cancer metastasis model using whole-genome promoter tiling microarrays.
Breast Cancer Res. 2008; 10(4):R62 [PubMed] Free Access to Full Article Related Publications
INTRODUCTION: Breast cancer metastasis is a complex, multi-step biological process. Genetic mutations along with epigenetic alterations in the form of DNA methylation patterns and histone modifications contribute to metastasis-related gene expression changes and genomic instability. So far, these epigenetic contributions to breast cancer metastasis have not been well characterized, and there is only a limited understanding of the functional mechanisms affected by such epigenetic alterations. Furthermore, no genome-wide assessments have been undertaken to identify altered DNA methylation patterns in the context of metastasis and their effects on specific functional pathways or gene networks.
METHODS: We have used a human gene promoter tiling microarray platform to analyze a cell line model of metastasis to lymph nodes composed of a poorly metastatic MDA-MB-468GFP human breast adenocarcinoma cell line and its highly metastatic variant (468LN). Gene networks and pathways associated with metastasis were identified, and target genes associated with epithelial-mesenchymal transition were validated with respect to DNA methylation effects on gene expression.
RESULTS: We integrated data from the tiling microarrays with targets identified by Ingenuity Pathways Analysis software and observed epigenetic variations in genes implicated in epithelial-mesenchymal transition and with tumor cell migration. We identified widespread genomic hypermethylation and hypomethylation events in these cells and we confirmed functional associations between methylation status and expression of the CDH1, CST6, EGFR, SNAI2 and ZEB2 genes by quantitative real-time PCR. Our data also suggest that the complex genomic reorganization present in cancer cells may be superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes.
CONCLUSION: This is the first whole-genome approach to identify genome-wide and gene-specific epigenetic alterations, and the functional consequences of these changes, in the context of breast cancer metastasis to lymph nodes. This approach allows the development of epigenetic signatures of metastasis to be used concurrently with genomic signatures to improve mapping of the evolving molecular landscape of metastasis and to permit translational approaches to target epigenetically regulated molecular pathways related to metastatic progression.

Qiu J, Ai L, Ramachandran C, et al.
Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas.
Lab Invest. 2008; 88(9):910-25 [PubMed] Free Access to Full Article Related Publications
DNA hypermethylation-mediated gene silencing is a frequent and early contributor to aberrant cell growth and invasion in cancer. Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Morbidity and mortality are high in glioma patients because tumors are resistant to treatment and are highly invasive into surrounding brain tissue rendering complete surgical resection impossible. Invasiveness is regulated by the interplay between secreted proteases (eg, cathepsins) and their endogenous inhibitors (cystatins). In our previous studies we identified cystatin E/M (CST6) as a frequent target of epigenetic silencing in glioma. Cystatin E/M is a potent inhibitor of cathepsin B, which is frequently overexpressed in glioma. Here, we study the expression of cystatin E/M in normal brain and show that it is highly and moderately expressed in oligodendrocytes and astrocytes, respectively, but not in neurons. Consistent with this, the CST6 promoter is hypomethylated in all normal samples using methylation-specific PCR, bisulfite genomic sequencing, and pyrosequencing. In contrast, 78% of 28 primary brain tumors demonstrated reduced/absent cystatin E/M expression using a tissue microarray and this reduced expression correlated with CST6 promoter hypermethylation. Interestingly, CST6 was expressed in neural stem cells (NSC) and markedly induced upon differentiation, whereas a glioma tumor initiating cell (TIC) line was completely blocked for CST6 expression by promoter methylation. Analysis of primary pediatric brain tumor-derived lines also showed CST6 downregulation and methylation in nearly 100% of 12 cases. Finally, ectopic expression of cystatin E/M in glioma lines reduced cell motility and invasion. These results demonstrate that epigenetic silencing of CST6 is frequent in adult and pediatric brain tumors and occurs in TICs, which are thought to give rise to the tumor. CST6 methylation may therefore represent a novel prognostic marker and therapeutic target specifically altered in TICs.

Veena MS, Lee G, Keppler D, et al.
Inactivation of the cystatin E/M tumor suppressor gene in cervical cancer.
Genes Chromosomes Cancer. 2008; 47(9):740-54 [PubMed] Free Access to Full Article Related Publications
We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5'aza 2-deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation.

Roll JD, Rivenbark AG, Jones WD, Coleman WB
DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines.
Mol Cancer. 2008; 7:15 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines.
RESULTS: The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A. This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers.
CONCLUSION: These observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers.

Luo X, Pan Q, Liu L, Chegini N
Genomic and proteomic profiling II: comparative assessment of gene expression profiles in leiomyomas, keloids, and surgically-induced scars.
Reprod Biol Endocrinol. 2007; 5:35 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Leiomyoma have often been compared to keloids because of their fibrotic characteristic and higher rate of occurrence among African Americans as compared to other ethnic groups. To evaluate such a correlation at molecular level this study comparatively analyzed leiomyomas with keloids, surgical scars and peritoneal adhesions to identify genes that are either commonly and/or individually distinguish these fibrotic disorders despite differences in the nature of their development and growth.
METHODS: Microarray gene expression profiling and realtime PCR.
RESULTS: The analysis identified 3 to 12% of the genes on the arrays as differentially expressed among these tissues based on P ranking at greater than or equal to 0.005 followed by 2-fold cutoff change selection. Of these genes about 400 genes were identified as differentially expressed in leiomyomas as compared to keloids/incisional scars, and 85 genes as compared to peritoneal adhesions (greater than or equal to 0.01). Functional analysis indicated that the majority of these genes serve as regulators of cell growth (cell cycle/apoptosis), tissue turnover, transcription factors and signal transduction. Of these genes the expression of E2F1, RUNX3, EGR3, TBPIP, ECM-2, ESM1, THBS1, GAS1, ADAM17, CST6, FBLN5, and COL18A was confirmed in these tissues using quantitative realtime PCR based on low-density arrays.
CONCLUSION: the results indicated that the molecular feature of leiomyomas is comparable but may be under different tissue-specific regulatory control to those of keloids and differ at the levels rather than tissue-specific expression of selected number of genes functionally regulating cell growth and apoptosis, inflammation, angiogenesis and tissue turnover.

Pan Q, Luo X, Chegini N
Genomic and proteomic profiling I: leiomyomas in African Americans and Caucasians.
Reprod Biol Endocrinol. 2007; 5:34 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Clinical observations indicate that leiomyomas occur more frequently in African Americans compared to other ethnic groups with unknown etiology. To identify the molecular basis for the difference we compared leiomyomas form A. Americans with Caucasians using genomic and proteomic strategies.
METHODS: Microarray, realtime PCR, 2D-PAGE, mass spectrometry, Western blotting and immunohistochemistry.
RESULTS: Using Affymetrix U133A array and analysis based on P ranking (P < 0.01) 1470 genes were identified as differentially expressed in leiomyomas compared to myometrium regardless of ethnicity. Of these, 268 genes were either over-expressed (177 genes) or under-expressed (91 genes) based on P < 0.01 followed by 2-fold cutoff selection in leiomyomas of A. Americans as compared to Caucasians. Among them, the expression E2F1, RUNX3, EGR3, TBPIP, ECM2, ESM1, THBS1, GAS1, ADAM17, CST6, CST7, FBLN5, ICAM2, EDN1 and COL18 was validated using realtime PCR low-density arrays. 2D PAGE coupled with image analysis identified 332 protein spots of which the density/volume of 31 varied by greater than or equal to 1.5 fold in leiomyomas as compared to myometrium. The density/volume of 34 protein-spots varied by greater than or equal to 1.5 fold (26 increased and 8 decreased) in leiomyomas of A. Americans as compared to Caucasians. Tandem mass spectrometric analysis of 15 protein spots identified several proteins whose transcripts were also identified by microarray, including 14-3-3 beta and mimecan, whose expression was confirmed using western blotting and immunohistochemistry.
CONCLUSION: These findings imply that the level rather than the ethnic-specific expression of a number of genes and proteins may account for the difference between leiomyomas and possibly myometrium, in A. Americans and Caucasians. Further study using larger sample size is required to confirm these findings.

Rivenbark AG, Livasy CA, Boyd CE, et al.
Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions.
Exp Mol Pathol. 2007; 83(2):188-97 [PubMed] Free Access to Full Article Related Publications
CST6 is a breast tumor suppressor gene that is expressed in normal breast epithelium, but is epigenetically silenced as a consequence of promoter hypermethylation in metastatic breast cancer cell lines. In the current study, we investigated the expression and methylation status of CST6 in primary breast tumors and lymph node metastases. 25/45 (56%) primary tumors and 17/20 (85%) lymph node metastases expressed significantly lower levels of cystatin M compared to normal breast tissue. Bisulfite sequencing demonstrated CST6 promoter hypermethylation in 11/23 (48%) neoplastic lesions analyzed, including 3/11 (27%) primary tumors and 8/12 (67%) lymph node metastases. In most cases (12/23, 52%), the expression of cystatin M directly reflected CST6 promoter methylation status. In remaining lesions (8/23, 35%) loss of cystatin M was not associated with CST6 promoter hypermethylation, indicating that other mechanisms can account for loss of CST6 expression. These results show that methylation-dependent silencing of CST6 occurs in a subset of primary breast cancers, but more frequently in metastatic lesions, possibly reflecting progression-related genomic events. To examine this possibility, primary breast tumors and matched lymph node metastases were analyzed. In 2/3 (67%) patients, primary tumors were positive for cystatin M and negative for CST6 promoter methylation, and matched metastatic lesions lacked cystatin M expression and CST6 was hypermethylated. This observation suggests that progression-related epigenetic alterations in CST6 gene expression can accompany metastatic spread from a primary tumor site. Overall, the results of the current investigation suggest that methylation-dependent epigenetic silencing of CST6 represents an important mechanism for loss of CST6 during breast tumorigenesis and/or progression to metastasis.

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