pubmed > ESR1 > cdh1

Antitumoral activity of liraglutide, a new DNMT inhibitor in breast cancer cells in vitro and in vivo.
Breast cancer (BC) is the most frequently diagnosed female cancer and second leading cause of death. Despite the discovery of many antineoplastic drugs for BC, the current therapy is not totally efficient. In this study, we investigated the potential of repurposing the well-known diabetes type II drug liraglutide to modulate epigenetic modifications in BC cells lines in vitro and in vivo via Ehrlich mice tumors models. The in vitro results revealed a significant reduction on cell viability, migration, DNMT activity and displayed lower levels of global DNA methylation in BC cell lines after liraglutide treatment. The interaction between liraglutide and the DNMT enzymes resulted in a decrease profile of DNA methylation for the CDH1, ESR1 and ADAM33 gene promoter regions and, consequently, increased their gene and protein expression levels. To elucidate the possible interaction between liraglutide and the DNMT1 protein, we performed an in silico study that indicates liraglutide binding in the catalytic cleft via hydrogen bonds and salt bridges with the interdomain contacts and disturbs the overall enzyme conformation. The in vivo study was also able to reveal that liraglutide and the combined treatment of liraglutide and paclitaxel or methotrexate were effective in reducing tumor growth. Moreover, the modulation of CDH1 and ADAM33 mouse gene expression by DNA demethylation suggests a role for liraglutide in DNMT activity in vivo. Altogether, these results indicate that liraglutide may be further analysed as a new adjuvant treatment for BC.
Publication Date: 2021-09-18
Journal: Chemico-biological interactions

DNA Methylation as a Diagnostic Biomarker for Malignant Mesothelioma: A Systematic Review and Meta-Analysis.
Malignant mesothelioma is an aggressive cancer type linked to asbestos exposure. Because of several intrinsic challenges, mesothelioma is often diagnosed in an advanced disease stage. Therefore, there is a need for diagnostic biomarkers that may contribute to early detection. Recently, the epigenome of tumors is being extensively investigated to identify biomarkers. This manuscript is a systematic review summarizing the state-of-the-art research investigating DNA methylation in mesothelioma. Four literature databases (PubMed, Scopus, Web of Science, MEDLINE) were systematically searched for studies investigating DNA methylation in mesothelioma up to October 16, 2020. A meta-analysis was performed per gene investigated in at least two independent studies. A total of 53 studies investigated DNA methylation of 97 genes in mesothelioma and are described in a qualitative overview. Furthermore, ten studies investigating 13 genes (APC, CDH1, CDKN2A, DAPK, ESR1, MGMT, miR-34b/c, PGR, RARβ, RASSF1, SFRP1, SFRP4, WIF1) were included in the quantitative meta-analysis. In this meta-analysis, the APC gene is significantly hypomethylated in mesothelioma, whereas CDH1, ESR1, miR-34b/c, PGR, RARβ, SFRP1, and WIF1 are significantly hypermethylated in mesothelioma. The three genes that are the most appropriate candidate biomarkers from this meta-analysis are APC, miR-34b/c, and WIF1. Nevertheless, both study number and study objects comprised in this meta-analysis are too low to draw final conclusions on their clinical applications. The elucidation of the genome-wide DNA methylation profile of mesothelioma is desirable in the future, using a standardized genome-wide methylation analysis approach. The most informative CpG sites from this signature could then form the basis of a panel of highly sensitive and specific biomarkers that can be used for the diagnosis of mesothelioma and even for the screening of an at high-risk population of asbestos-exposed individuals.
Publication Date: 2021-06-04
Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Network Pharmacology Interpretation of Fuzheng-Jiedu Decoction against Colorectal Cancer.
Traditional Chinese medicine (TCM) believes that the pathogenic factors of colorectal cancer (CRC) are "deficiency, dampness, stasis, and toxin," and Fuzheng-Jiedu Decoction (FJD) can resist these factors. In this study, we want to find out the potential targets and pathways of FJD in the treatment of CRC and also explain from a scientific point of view that FJD multidrug combination can resist "deficiency, dampness, stasis, and toxin." We get the composition of FJD from the TCMSP database and get its potential target. We also get the potential target of colorectal cancer according to the OMIM Database, TTD Database, GeneCards Database, CTD Database, DrugBank Database, and DisGeNET Database. Subsequently, PPI analysis, KEGG pathways analysis, and GO biological processes analysis were carried out for the target of FJD in the therapy of colorectal cancer. In addition, we have also built a relevant network diagram. In this study, we identified four core compounds of FJD in the therapy of colorectal cancer, including quercetin, kaempferol, beta-sitosterol, and stigmasterol. At the same time, we also obtained 30 core targets, including STAT3, INS, TP53, VEGFA, AKT1, TNF, IL6, JUN, EGF, CASP3, MAPK3, MAPK1, MAPK8, SRC, IGF1, CCND1, ESR1, EGFR, PTEN, MTOR, FOS, PTGS2, CXCL8, HRAS, CDH1, BCL2L1, FN1, MMP9, ERBB2, and JAK2. FJD treatment of colorectal cancer mainly involves 112 KEGG pathways, including FoxO (hsa04068) signaling pathway, PI3K-Akt (hsa04151) signaling pathway, HIF-1 (hsa04066) signaling pathway, T cell receptor (hsa04660) signaling pathway, and ErbB (hsa04012) signaling pathway. At the same time, 330 GO biological processes were summarized, including cell proliferation, cell apoptosis, angiogenesis, inflammation, and immune. In this study, we found that FJD can regulate cell proliferation, apoptosis, inflammation and immunity, and angiogenesis through PI3K-Akt signaling pathway to play an anti-CRC effect.
Publication Date: 2021-03-11
Journal: Evidence-based complementary and alternative medicine : eCAM

Cellular Mechanisms Triggered by the Cotreatment of Resveratrol and Doxorubicin in Breast Cancer: A Translational In Vitro-In Silico Model.
Doxorubicin (Doxo) is the most effective chemotherapeutic agent for the treatment of breast cancer. However, resistance to Doxo is common. Adjuvant compounds capable of modulating mechanisms involved in Doxo resistance may potentiate the effectiveness of the drug. Resveratrol (Rsv) has been tested as an adjuvant in mammary malignancies. However, the cellular and molecular mechanisms underlying the effects of cotreatment with Doxo and Rsv in breast cancer are poorly understood. Here, we combined in vitro and in silico analysis to characterize these mechanisms. In vitro, we employed a clinically relevant experimental design consisting of acute (24 h) treatment followed by 15 days of analysis. Acute Rsv potentiated the long-lasting effect of Doxo through the induction of apoptosis and senescence. Cells that survived to the cotreatment triggered high levels of autophagy. Autophagy inhibition during its peak of activation but not concomitant with Doxo+Rsv increased the long-term toxicity of the cotreatment. To uncover key proteins potentially associated with in vitro effects, an in silico multistep strategy was implemented. Chemical-protein networks were predicted based on constitutive gene expression of MCF7 cells and interatomic data from breast cancer. Topological analysis, KM survival analysis, and a quantitative model based on the connectivity between apoptosis, senescence, and autophagy were performed. We found seven putative genes predicted to be modulated by Rsv in the context of Doxo treatment: CCND1, CDH1, ESR1, HSP90AA1, MAPK3, PTPN11, and RPS6KB1. Six out of these seven genes have been experimentally proven to be modulated by Rsv in cancer cells, with 4 of the 6 genes in MCF7 cells. In conclusion, acute Rsv potentiated the long-term toxicity of Doxo in breast cancer potentially through the modulation of genes and mechanisms involved in Doxo resistance. Rational autophagy inhibition potentiated the effects of Rsv+Doxo, a strategy that should be further tested in animal models.
Publication Date: 2020-11-19
Journal: Oxidative medicine and cellular longevity

Prognostic Biomarkers in Endometrial Cancer: A Systematic Review and Meta-Analysis.
Endometrial cancer (EC) is the sixth most common cancer in women worldwide and its mortality is directly associated with the presence of poor prognostic factors driving tumor recurrence. Stratification systems are based on few molecular, and mostly clinical and pathological parameters, but these systems remain inaccurate. Therefore, identifying prognostic EC biomarkers is crucial for improving risk assessment pre- and postoperatively and to guide treatment decisions. This systematic review gathers all protein biomarkers associated with clinical prognostic factors of EC, recurrence and survival. Relevant studies were identified by searching the PubMed database from 1991 to February 2020. A total number of 398 studies matched our criteria, which compiled 255 proteins associated with the prognosis of EC. MUC16, ESR1, PGR, TP53, WFDC2, MKI67, ERBB2, L1CAM, CDH1, PTEN and MMR proteins are the most validated biomarkers. On the basis of our meta-analysis ESR1, TP53 and WFDC2 showed potential usefulness for predicting overall survival in EC. Limitations of the published studies in terms of appropriate study design, lack of high-throughput measurements, and statistical deficiencies are highlighted, and new approaches and perspectives for the identification and validation of clinically valuable EC prognostic biomarkers are discussed.
Publication Date: 2020-06-21
Journal: Journal of clinical medicine

Genetic alterations and their association with clinicopathologic characteristics in advanced breast carcinomas: focusing on clinically actionable genetic alterations.
Breast carcinomas (BCs) are genetically heterogeneous and associated with numerous mutations which can be used to predict outcomes and initiate targeted therapies. We investigated clinicopathologic characteristics associated with gene mutations detected using the FoundationOne CDx assay in a cohort of 223 clinically advanced BCs (66 locally recurrent and 157 metastatic) from our institution. One hundred fifty unique mutations were identified (total 1008) in the cohort, with the most prevalent (>10%) including TP53 (53.8%), PIK3CA (35%), MYC (22%), CCND1 (19.7%), FGF19 (19.7%), FGF4 (16.6%), FGF3 (16.1%), ZNF703 (14.8%), ESR1 (13.9%), FGFR1 (13.5%), PTEN (12.1%), and CDH1 (10.8%). ERBB2 genetic alteration was most common in human epidermal growth factor receptor 2 (HER2)-positive BCs, and GATA3 and ESR1 mutations were only identified in hormone receptor-positive BC. Mutations enriched in triple-negative BCs (TNBCs) included TP53, PTEN, RB1, and CDKN2A/B. CDH1 mutation was predominantly found in lobular carcinomas, and PIK3CA mutation was also enriched. Mutations enriched in metaplastic carcinomas with heterologous mesenchymal differentiation included TP53, PTEN, MCL1, CDKN2A/B, and NOTCH2. An increase in mutations of CCND1, FGF19, FGF4, FGF3, ESR1, and EMSY was identified in metastatic BCs compared with locally recurrent BCs. Overall, PIK3CA was the most frequent clinically actionable genetic alteration (35%), followed by MYC (22%), CCND1 (19.7%), and FGF3/FGF4/FGFR1 (16%). In conclusion, our study provides genetic insight into the biology of advanced BCs and summarizes their most frequent clinically actionable genetic alterations, generating useful genomic information for potential improvement of patient management.
Publication Date: 2020-05-24
Journal: Human pathology

Association of a novel circulating tumor DNA next-generating sequencing platform with circulating tumor cells (CTCs) and CTC clusters in metastatic breast cancer.
Liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), can be used to understand disease prognosis, tumor heterogeneity, and dynamic response to treatment in metastatic breast cancer (MBC). We explored a novel, 180-gene ctDNA panel and the association of this platform with CTCs and CTC clusters. A total of 40 samples from 22 patients with MBC were included in the study. For the primary analysis, all patients had ctDNA sequencing using the PredicinePLUS™ platform. CTCs and CTC clusters were examined using the CellSearch™ System. Clinical and pathological variables were reported using descriptive analyses. Associations between CTC count and specific genomic alterations were tested using the Mann-Whitney U test. Of 43 sequenced patients, 40 (93%) had at least one detectable genomic alteration with a median of 6 (range 1-22). Fifty-seven different genes were altered, and the landscape of genomic alterations was representative of MBC, including the commonly encountered alterations TP53, PTEN, PIK3CA, ATM, BRCA1, CCND1, ESR1, and MYC. In patients with predominantly hormone-receptor-positive MBC, the number of CTCs was significantly associated with alterations in ESR1 (P < 0.005), GATA3 (P < 0.05), CDH1 (P < 0.0005), and CCND1 (P < 0.05) (Mann-Whitney U test). Thirty-six percent of patients had CTC clusters, which were associated with alterations in CDH1, CCND1, and BRCA1 (all P < 0.05, Mann-Whitney U test). In an independent validation cohort, CTC enumeration confirmed significant associations with ESR1 and GATA3, while CTC clusters were significantly associated with CDH1. We report on a novel ctDNA platform that detected genomic alterations in the vast majority of tested patients, further indicating potential clinical utility for capturing disease heterogeneity and for disease monitoring. Detection of CTCs and CTC clusters was associated with particular genomic profiles.
Publication Date: 2019-12-06
Journal: Breast cancer research : BCR

Targeted next-generation sequencing identifies clinically relevant somatic mutations in a large cohort of inflammatory breast cancer.
Inflammatory breast cancer (IBC) is the most aggressive form of primary breast cancer. Using a custom-made breast cancer gene sequencing panel, we investigated somatic mutations in IBC to better understand the genomic differences compared with non-IBC and to consider new targeted therapy in IBC patients. Targeted next-generation sequencing (NGS) of 91 candidate breast cancer-associated genes was performed on 156 fresh-frozen breast tumor tissues from IBC patients. Mutational profiles from 197 primary breast tumors from The Cancer Genome Atlas (TCGA) were used as non-IBC controls for comparison analysis. The mutational landscape of IBC was correlated with clinicopathological data and outcomes. After genotype calling and algorithmic annotations, we identified 392 deleterious variants in IBC and 320 variants in non-IBC cohorts, respectively. IBC tumors harbored more mutations than non-IBC (2.5 per sample vs. 1.6 per sample, p < 0.0001). Eighteen mutated genes were significantly different between the two cohorts, namely TP53, CDH1, NOTCH2, MYH9, BRCA2, ERBB4, POLE, FGFR3, ROS1, NOTCH4, LAMA2, EGFR, BRCA1, TP53BP1, ESR1, THBS1, CASP8, and NOTCH1. In IBC, the most frequently mutated genes were TP53 (43.0%), PIK3CA (29.5%), MYH9 (8.3%), NOTCH2 (8.3%), BRCA2 (7.7%), ERBB4 (7.1%), FGFR3 (6.4%), POLE (6.4%), LAMA2 (5.8%), ARID1A (5.1%), NOTCH4 (5.1%), and ROS1 (5.1%). After grouping 91 genes on 10 signaling pathways, we found that the DNA repair pathway for the triple-negative breast cancer (TNBC) subgroup, the RTK/RAS/MAPK and cell cycle pathways for the HR Breast cancer-specific targeted NGS uncovered a high frequency of deleterious somatic mutations in IBC, some of which may be relevant for clinical management.
Publication Date: 2018-08-09
Journal: Breast cancer research : BCR

Mixed ductal-lobular carcinomas: evidence for progression from ductal to lobular morphology.
Mixed ductal-lobular carcinomas (MDLs) show both ductal and lobular morphology, and constitute an archetypal example of intratumoural morphological heterogeneity. The mechanisms underlying the coexistence of these different morphological entities are poorly understood, although theories include that these components either represent 'collision' of independent tumours or evolve from a common ancestor. We performed comprehensive clinicopathological analysis of a cohort of 82 MDLs, and found that: (1) MDLs more frequently coexist with ductal carcinoma in situ (DCIS) than with lobular carcinoma in situ (LCIS); (2) the E-cadherin-catenin complex was normal in the ductal component in 77.6% of tumours; and (3) in the lobular component, E-cadherin was almost always aberrantly located in the cytoplasm, in contrast to invasive lobular carcinoma (ILC), where E-cadherin is typically absent. Comparative genomic hybridization and multiregion whole exome sequencing of four representative cases revealed that all morphologically distinct components within an individual case were clonally related. The mutations identified varied between cases; those associated with a common clonal ancestry included BRCA2, TBX3, and TP53, whereas those associated with clonal divergence included CDH1 and ESR1. Together, these data support a model in which separate morphological components of MDLs arise from a common ancestor, and lobular morphology can arise via a ductal pathway of tumour progression. In MDLs that present with LCIS and DCIS, the clonal divergence probably occurs early, and is frequently associated with complete loss of E-cadherin expression, as in ILC, whereas, in the majority of MDLs, which present with DCIS but not LCIS, direct clonal divergence from the ductal to the lobular phenotype occurs late in tumour evolution, and is associated with aberrant expression of E-cadherin. The mechanisms driving the phenotypic change may involve E-cadherin-catenin complex deregulation, but are yet to be fully elucidated, as there is significant intertumoural heterogeneity, and each case may have a unique molecular mechanism. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Publication Date: 2018-01-19
Journal: The Journal of pathology

Mutation screening of 10 cancer susceptibility genes in unselected breast cancer patients.
Variants of cancer susceptibility genes other than BRCA1/2 have been proved to be associated with increased risks of breast cancer. This study was performed to investigate the spectrum and prevalence of mutations in 10 cancer susceptibility genes in paired tumor/normal tissues of 292 unselected Chinese breast cancer patients. We performed an analysis of germline and somatic variants in ATM, CDH1, CHEK2, ESR1, GATA3, MAP3K1, MSH2, PALB2, RB1 and STK11 genes by integrating microfluidic PCR-based target enrichment and next-generation sequencing technologies. In total, 3 germline and 25 somatic deleterious mutations were found among 27 patients (9.25%), and 17 of them were novel mutations. Most deleterious mutations were prevalent in luminal A invasive breast cancer (P = .014). We also observed 83 variants of uncertain significance (VUS) in 100 patients (34.25%), 23 of which were predicted to be deleterious by in silico prediction programs (MetaSVM and MetaLR). VUS carriers had higher positive rate of lymph node metastasis than non-carriers (P = .008) and were predominantly present in ER+ tumors (P = .018). Our findings would enhance the understanding of the molecular mechanisms of breast cancer in Chinese population.
Publication Date: 2017-06-06
Journal: Clinical genetics

Mutational Profile of Metastatic Breast Cancers: A Retrospective Analysis.
Major advances have been achieved in the characterization of early breast cancer (eBC) genomic profiles. Metastatic breast cancer (mBC) is associated with poor outcomes, yet limited information is available on the genomic profile of this disease. This study aims to decipher mutational profiles of mBC using next-generation sequencing. Whole-exome sequencing was performed on 216 tumor-blood pairs from mBC patients who underwent a biopsy in the context of the SAFIR01, SAFIR02, SHIVA, or Molecular Screening for Cancer Treatment Optimization (MOSCATO) prospective trials. Mutational profiles from 772 primary breast tumors from The Cancer Genome Atlas (TCGA) were used as a reference for comparing primary and mBC mutational profiles. Twelve genes (TP53, PIK3CA, GATA3, ESR1, MAP3K1, CDH1, AKT1, MAP2K4, RB1, PTEN, CBFB, and CDKN2A) were identified as significantly mutated in mBC (false discovery rate [FDR] < 0.1). Eight genes (ESR1, FSIP2, FRAS1, OSBPL3, EDC4, PALB2, IGFN1, and AGRN) were more frequently mutated in mBC as compared to eBC (FDR < 0.01). ESR1 was identified both as a driver and as a metastatic gene (n = 22, odds ratio = 29, 95% CI [9-155], p = 1.2e-12) and also presented with focal amplification (n = 9) for a total of 31 mBCs with either ESR1 mutation or amplification, including 27 hormone receptor positive (HR+) and HER2 negative (HER2-) mBCs (19%). HR+/HER2- mBC presented a high prevalence of mutations on genes located on the mechanistic target of rapamycin (mTOR) pathway (TSC1 and TSC2) as compared to HR+/HER2- eBC (respectively 6% and 0.7%, p = 0.0004). Other actionable genes were more frequently mutated in HR+ mBC, including ERBB4 (n = 8), NOTCH3 (n = 7), and ALK (n = 7). Analysis of mutational signatures revealed a significant increase in APOBEC-mediated mutagenesis in HR+/HER2- metastatic tumors as compared to primary TCGA samples (p < 2e-16). The main limitations of this study include the absence of bone metastases and the size of the cohort, which might not have allowed the identification of rare mutations and their effect on survival. This work reports the results of the analysis of the first large-scale study on mutation profiles of mBC. This study revealed genomic alterations and mutational signatures involved in the resistance to therapies, including actionable mutations.
Publication Date: 2016-12-28
Journal: PLoS medicine

Role of heme oxygenase-1 in demethylating effects on SKM-1 cells induced by decitabine.
We evaluated the influence of heme oxygenase-1 (HO-1) gene inhibition in myelodysplastic syndrome (MDS) cell line SKM-1 on enhancement of the demethylating effects of decitabine on p15, and explored the possible mechanism. DNMT1 gene expression in SKM-1 cells was silenced by being transfected by a constructed siRNA with liposomes. The proliferation inhibition rates after drug treatment were detected by cell counting kit-8 assay. The apoptotic rates were detected by Annexin V/PI assay with flow cytometry. The expressions of p16, p15, TP73, CDH1, ESR1, and PDLIM4 mRNAs were detected by real-time PCR, and those of HO-1, DNMT1, DNMT3A, DNMT3B, HDAC, and p15 proteins were measured by western blot. The degree of methylation of the p15 gene was analyzed by using methylation-specific PCR (MSP). CCK-8 assay showed that after HO-1 gene expression was inhibited; the proliferation rate of SKM-1 cells treated by decitabine (70.91 ± 0.05%) was significantly higher than that of the control group (53.67 ± 0.05%). Flow cytometry showed that the apoptotic rate of SKM- 1 cells treated by decitabine in combination with HO-1 expression inhibition (44.25 ± 0.05%) exceeded that of the cells treated by this drug alone (37.70 ± 0.05%). MSP showed that inhibiting HO-1 expression significantly increased the degree of methylation of the p15 gene. As suggested by western blot, the degree of methylation of the p15 protein was changed after decitabine treatment when the expression of the HO-1 protein was changed, being associated with the affected DNMT1 expression. Inhibited HO-1 expression attenuated the hypermethylation of CDKN2B by suppressing DNMT1, which was conducive to treatment by cooperating with decitabine. In conclusion, the findings of this study provide valuable experimental evidence for targeted MDS therapy, and a theoretical basis for further studies.
Publication Date: 2016-01-20
Journal: Genetics and molecular research : GMR

Sera DNA Methylation of CDH1, DNMT3b and ESR1 Promoters as Biomarker for the Early Diagnosis of Hepatitis B Virus-Related Hepatocellular Carcinoma.
DNA methylation mainly affects tumor suppressor genes in the development of hepatocellular carcinoma (HCC). However, sera methylation of specific genes in hepatitis B virus (HBV)-related HCC remains unknown. The purpose of this study was to identify methylation frequencies of sera E-cadherin (CDH1), DNA methyltransferase 3b (DNMT3b) and estrogen receptor 1 (ESR1) promoter in HBV-related HCC and analyze the associated clinical significance. Methylation-specific PCR was used to determine the frequencies of DNA methylation for CDH1, DNMT3b and ESR1 genes in sera from 183 patients with HCC, 47 liver cirrhosis (LC), 126 chronic hepatitis B (CHB), and 50 normal controls (NCs). Significantly higher frequencies of methylation of CDH1, DNMT3b and ESR1 were found in HBV-related HCC compared with LC, CHB and NCs. Nodule numbers, tumor size and the presence of liver cirrhosis were significantly associated with gene methylation status in HBV-related HCC. Moreover, HBV may have a strong and enhanced effect on the concurrent methylation of CDH1, DNMT3b and ESR1 in HBV-related HCC. More importantly, combined methylation as a biomarker displayed significantly higher diagnostic value than AFP to discriminate HCC from CHB and LC. Aberrant sera DNA methylation of CDH1, DNMT3b and ESR1 gene promoters could be a biomarker in the early diagnosis of HBV-related HCC.
Publication Date: 2015-12-15
Journal: Digestive diseases and sciences

Selective Inhibition of SIN3 Corepressor with Avermectins as a Novel Therapeutic Strategy in Triple-Negative Breast Cancer.
Triple-negative breast cancers (TNBC) lacking estrogen, progesterone, and HER2 receptors account for 10% to 20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a pressing unmet clinical need. We previously identified a molecularly targeted approach to target aberrant epigenetics of TNBC using a peptide corresponding to the SIN3 interaction domain (SID) of MAD. SID peptide selectively blocked binding of SID-containing proteins to the paired α-helix (PAH2) domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial-mesenchymal transition (EMT). To find small molecule inhibitor (SMI) mimetics of SID peptide, we performed an in silico screen for PAH2 domain-binding compounds. This led to the identification of the avermectin macrocyclic lactone derivatives selamectin and ivermectin (Mectizan) as candidate compounds. Both selamectin and ivermectin phenocopied the effects of SID peptide to block SIN3-PAH2 interaction with MAD, induce expression of CDH1 and ESR1, and restore tamoxifen sensitivity in MDA-MB-231 human and MMTV-Myc mouse TNBC cells in vitro. Treatment with selamectin or ivermectin led to transcriptional modulation of genes associated with EMT and maintenance of a cancer stem cell phenotype in TNBC cells. This resulted in impairment of clonogenic self-renewal in vitro and inhibition of tumor growth and metastasis in vivo. Underlining the potential of avermectins in TNBC, pathway analysis revealed that selamectin also modulated the expression of therapeutically targetable genes. Consistent with this, an unbiased drug screen in TNBC cells identified selamectin-induced sensitization to a number of drugs, including those targeting modulated genes.
Publication Date: 2015-06-17
Journal: Molecular cancer therapeutics

[Numerical impairments in genes in breast cancer: a multiplex ligation-dependent probe amplification study].
To analyze breast cancer samples using the new technique multiplex ligation-dependent probe amplification (MLPA) assay. MATERIAL AND METHODS; Formalin-fixed paraffin-embedded breast carcinoma samples from 65 patients were examined. After manual microdissection, DNA was isolated using a commercial kit ("QIAGEN") and analyzed with SALSA MLPA KIT P078-B1 Breast Tumour ("MRC-Holland"). Capillary electrophoresis provided results. MLPA assay was successful in all examined samples. The amplification and deletion frequencies of the analyzed genes were in line with the literature data. The technique requires conventional work-related skills in a molecular genetic laboratory and, as a whole, presents no problems with its usage. The interpretation of results is devoid of subjective meaning due to exclusively their mathematical analysis. MLPA assay provides an insight into numerical impairments in the following genes: ERBB2, MYC, TRAF4, C11orf30 (EMSY), ADAM9, IKBKB, CCNE1, TOP2A, CDH1, CDC6, ESR1, CPD, EGFR, MTDH, CCND1, BIRC5, MED1, FGFR1, MAPT, PRDM14, and AURKA. MLPA is an easy-to-use and promising method for multiplex genetic analysis of tumor cells in breast cancer.
Publication Date: 2014-10-14
Journal: Arkhiv patologii

Analysis of gene copy number alterations by multiplex ligation-dependent probe amplification in columnar cell lesions of the breast.
Columnar cell lesions (CCLs) are possible precursors of breast cancer, but little is known about the role of breast cancer-related genes in the progression of CCL to invasive breast cancer. Gene copy numbers of 17 breast cancer-related genes were analyzed using Multiplex Ligation-dependent Probe Amplification (MLPA) in CCL (N = 28), ductal carcinoma in situ (DCIS) grade I likely originating from CCL (N = 5), and paired CCL (N = 14/28) with DCIS (N = 7) and/or invasive carcinoma (N = 13). The genes included were BIRC5, C11orf30, CCND1, CCNE1, CDH1, CPD, EGFR, ERBB2, ESR1, FGFR1, IKBKB, MAPT, MED1, MTDH, MYC, TOP2A and TRAF4. No high level gene amplifications were observed in CCL, but copy number gains were encountered for the C11orf30 (3/28), MYC, CPD, MTDH (2/28), and CCND1, CCNE1, ESR1 and TOP2A genes (1/28). In addition, CDH1 showed loss in 2/28 and TOP2A in 1/28 cases. CCLs with or without atypia exhibited comparable numbers of copy number changes (p = 0.312). Overall, the frequency of gene copy number changes increased from CCL towards DCIS and invasive carcinoma (p = 0.004). Also in the cases with synchronous lesions, the CCLs exhibited fewer copy number changes than the DCIS/invasive carcinomas. CCLs carry copy number changes of several known breast cancer-related genes, thereby substantiating their role in breast carcinogenesis. Among them, CCND1 and ESR1 copy number gains and CDH1 copy number losses are of particular interest. Since the copy number changes observed were more prevalent in DCIS and invasive carcinoma than in CCL, the corresponding gene alterations may represent rather late occurring events in low nuclear grade breast carcinogenesis.
Publication Date: 2014-04-03
Journal: Cellular oncology (Dordrecht)

Dysregulation of microRNA expression drives aberrant DNA hypermethylation in basal-like breast cancer.
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.
Publication Date: 2013-12-04
Journal: International journal of oncology

Dysregulation of the epigenome in triple-negative breast cancers: basal-like and claudin-low breast cancers express aberrant DNA hypermethylation.
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.
Publication Date: 2013-09-21
Journal: Experimental and molecular pathology

Evaluation of protein expression and DNA methylation profiles detected by pyrosequencing in invasive breast cancer.
Breast carcinoma is the most common cancer with high mortality caused by metastatic disease. New molecular biomarkers predicting the tumour's metastatic potential would therefore improve metastasis prevention and personalised care. The aim of the study was to investigate the relationship between DNA methylation levels in invasivity and metastasising associated genes with aberrant protein expression and also to evaluate whether a similar DNA methylation level is present in the tumour and circulating cell-free DNA for utilising plasma DNA methylation as prognostic biomarker. By using pyrosequencing, we analysed DNA methylation levels of 11 genes, namely APC, ADAM23, CXCL12, ESR1, PGR B, CDH1, RASSF1A, SYK, TIMP3, BRMS1 and SOCS1 in tumour, plasma and peripheral blood cells from 34 patients with primary breast cancer, as well as plasma and peripheral blood cells from 50 healthy controls. Simultaneously, the expression of related proteins in paraffin-embedded tumour samples was evaluated by immunohistochemistry. Statistical analysis was performed by SPSS statistics 15.0 software. Tumour DNA hypermethylation was found in most commonly methylated RASSF1A (71.9%), APC (55.9%), ADAM23 (38%) and CXCL12 (34.4%) genes with methylation levels up to 86, 86, 53 and 64 %, respectively. In tumours, significantly higher methylation levels were found in nine genes, compared with the patients´ peripheral blood cell DNA. Furthermore, in patients methylation levels in peripheral blood cell DNA were significantly higher than in controls in CXCL12, ESR1 and TIMP3 genes, but the values did not exceed 15%. On the other hand, no correlations were observed in patients between DNA methylation in tumours and cell-free plasma DNA. Moreover, in patients and controls nearly identical values of cumulative DNA methylation (43.6 % ± 20.1 vs. 43.7 % ± 15.0) were observed in plasma samples. A variable spectrum from high to none expressions presented in tumour tissues in all of the proteins evaluated, however in APC and CXCL12 genes a visible decreasing trend of mean DNA methylation level with increasing expression of the corresponding protein was observed. The DNA methylation profiles manifested in our group of breast carcinomas are cancer specific, but they are not the only cause that affects the silencing of evaluated genes and the decrease of relevant protein products. The clinical utility of DNA methylation testing in peripheral blood cell DNA for cancer diagnosis and therapy need to be further investigated.
Publication Date: 2013-08-03
Journal: Neoplasma

Endometriosis is characterized by a distinct pattern of histone 3 and histone 4 lysine modifications.
The histone modification patterns in endometriosis have not been fully characterized. This gap in knowledge results in a poor understanding of the epigenetic mechanisms (and potential therapeutic targets) at play. We aimed to (1) assess global acetylation status of histone 3 (H3) and histone 4 (H4), (2) measure levels of H3 and H4 lysine (K) acetylation and methylation, and (3) to identify histone acetylation patterns in promoter regions of candidate genes in tissues from patients and controls. Global and K-specific acetylation/methylation levels of histones were measured in 24 lesions, 15 endometrium from patients, and 26 endometrium from controls. Chromatin immunoprecipitation (ChIP)-polymerase chain reaction was used to determine the histone acetylation status of the promoter regions of candidate genes in tissues. The lesions were globally hypoacetylated at H3 (but not H4) compared to eutopic endometrium from controls. Lesions had significantly lower levels of H3K9ac and H4K16ac compared to eutopic endometrium from patients and controls. Tissues from patients were hypermethylated at H3K4, H3K9, and H3K27 compared to endometrium from controls. The ChIP analysis showed hypoacetylation of H3/H4 within promoter regions of candidate genes known to be downregulated in endometriosis (e.g., HOXA10, ESR1, CDH1, and p21 (WAF1/Cip1) ) in lesions versus control endometrium. The stereoidogenic factor 1 (SF1) promoter region was enriched for acetylated H3 and H4 in lesions versus control tissues, correlating with its reported high expression in lesions. This study describes the histone code of lesions and endometrium from patients with endometriosis and provides support for a possible role of histone modification in modulation of gene expression in endometriosis.
Publication Date: 2013-08-01
Journal: Reproductive sciences (Thousand Oaks, Calif.)