CpG promoter hypo-methylation and up-regulation of microRNA-190b in hormone receptor-positive breast cancer

Publisher's version (útgefin grein)Estrogen receptor-positive breast cancer is subdivided into subtypes LuminalA and LuminalB, based on different expression patterns. MicroRNA-190b has been reported to be up-regulated in estrogen receptor-positive breast cancers. In this study we aimed to investigate the role of CpG promoter methylation in regulating miR-190b expression and its impact on clinical presentation and prognosis. DNA methylation analysis for the promotor of microRNA-190b was performed by pyrosequencing 549 primary breast tumors, of which 62 were carriers of the BRCA2999del5 founder mutation, 71 proximal normal breast samples and 16 breast derived cell lines. MicroRNA-190b expression was analysed in 67 primary breast tumors, 14 paired normal breast samples and 16 breast derived cell lines. Tissue microarrays (TMAs) were available for ER (n = 436), PR (n = 436), HER-2 (N = 258) and Ki67 (n = 248). MiR-190b had reduced promoter methylation in estrogen receptor-positive breast cancers (P = 1.02e–12, Median values: ER+ 24.3, ER– 38.26) and miR-190b’s expression was up-regulated in a correlative manner (P = 1.83e–06, Spearman’s rho –0.62). Through breast cancer specific survival analysis, we demonstrated that LuminalA patients exhibiting miR-190b hypo-methylation had better survival than other patients (P = 0.034, HR = 0.29, 95% CI 0.09-0.91). We, furthermore, demonstrated that miR-190b hypo-methylation occurs less frequently in ER+ tumors from BRCA2999del5 mutation carriers than in non-mutated individuals (P = 0.038, Χ2 = 4.32, n = 335). Our results suggest that upregulation of miR-190b may occur through loss of promoter DNA methylation during the development of estrogen-receptor (ER) positive breast cancers, and that miR-190b hypo-methylation leads to increased breast cancer specific survival within the LuminalA- subtype but not LuminalB.This work was funded b y Gongum Saman (EAF), The Icelandic cancer society (TG), and Icelandic Centre for Research RANNIS grant ID # 141395Peer reviewe

Melflufen, a peptide‐conjugated alkylator, is an efficient anti‐neo‐plastic drug in breast cancer cell lines

Publisher's version (útgefin grein)Melphalan flufenamide (hereinafter referred to as “melflufen”) is a peptide-conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal-derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA-MB231. The tumorigenic D492HER2 and MDA-MB231 cells were more sensitive than normal-derived D492 cells when treated with melflufen. Compared to the commonly used anti-cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA-MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins ƴH2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti-neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.We thank Inovotion for their work and support in the CAM assays.Peer Reviewe

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

The CHD4 helicase is identified as a new component of the genome surveillance machinery in a proteomic screen for factors enriched on chromatin after ionizing radiation (see also related paper by Smeenk et al. in this issue)

CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked FilesDNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer.Methylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation.The ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20% CpG methylation was found to be statistically significantly associated with reduced survival (HR = 2.3; P = 0.012). By thresholding at the clinically relevant CpG methylation level (>20%), we find the incidence of ALKBH3 promoter methylation to be 5% (13 out of 265).ALKBH3 is a novel addition to the catalogue of DNA repair genes found inactivated in breast cancer. Our results underscore a link between defective alkylation repair and breast cancer which, additionally, is found in association with poor disease outcome.Icelandic Centre for Researc

MicroRNA-190b Targets RFWD3 in ER Positive Breast Cancer

Abstract Background: In the year 2020 breast cancer was the most common form of cancer. Roughly 70% of breast cancers are estrogen receptor positive. MicroRNA-190b has previously been reported to be up-regulated in estrogen receptor positive breast cancers. Our group has previously demonstrated that microRNA-190b is hypomethylated in ER+ breast cancers, potentially leading to its upregulation. Results: In this study, using data from the Cancer Genome Atlas, we confirm that microRNA-190b is overexpressed in breast cancer via differential expression analysis and show that high expression of microRNA-190b results in more favorable outcomes in Luminal A patients (HR=0.29, 95% CI 0.12-0.71, P value=0.0063). MicroRNA190b target analysis, using immunoprecipitation of biotin labelled microRNA-190b, followed by RNA sequencing, identified RFWD3 as one of microRNA-190b’s regulatory targets in estrogen receptor positive breast cancer. Survival analysis of RFWD3 showed that elevated levels result in poorer overall survival in Luminal A breast cancer patients (HR = 2.22, 95% CI 1.33-3.71, P = 0.002). Gene ontology analysis of our sequencing results indicate that miR-190b may have a role in breast cancer development and/or tumorigenesis and that it may be a suitable tool in characterization between the estrogen receptor positive subtypes, Luminal A and Luminal B.Conclusions: We show that miR-190b targets RFWD3 in ER+ breast cancers leading to lower RFWD3 protein expression. Low levels of RFWD3 are associated with better outcomes in Luminal A breast cancer patients but not in Luminal B patients.</jats:p

MicroRNA-190b Targets RFWD3 in Estrogen Receptor–Positive Breast Cancer

Background: In the year 2020, breast cancer was the most common form of cancer worldwide. Roughly 70% of breast cancers are estrogen receptor–positive (ER+). MicroRNA-190b (miR-190b) has previously been reported to be upregulated in ER+ breast cancers. Previously, we have demonstrated that miR-190b is hypomethylated in ER+ breast cancers, potentially leading to its upregulation. Objectives: To further study the role of miR-190b in ER+ breast cancer and to identify its clinically relevant targets in breast cancer. Design: Patient cohort and cell line–based RNA-sequencing analysis. Methods: The Cancer Genome Atlas was used to obtain gene expression data and clinical information on patients with breast cancer. To identify messenger RNA (mRNA) targets for miR-190b, the ER+ breast cancer cell line T-47D was used to immunoprecipitate biotin-labeled miR-190b followed by RNA sequencing. Western blot was used to confirm miR-190b target. Patient survival based on miR-190b and selected target was studied using the Cancer Genome Atlas. Results: In this study, we confirm that miR-190b is overexpressed in breast cancer via differential expression analysis and show that high expression of miR-190b results in more favorable outcomes in Luminal A patients, hazard ratio (HR) = 0.29, 95% confidence interval [CI] = 0.12-0.71, P  = .0063. MicroRNA-190b target analysis identified RING finger and WD repeat domain 3 (RFWD3) as one of miR-190b regulatory targets in ER+ breast cancer. Survival analysis of RFWD3 showed that elevated levels result in poorer overall survival in patients with Luminal A breast cancer (HR = 2.22, 95% CI = 1.33-3.71, P  = .002). Gene ontology analysis of our sequencing results indicates that miR-190b may have a role in breast cancer development and/or tumorigenesis and that it may be a suitable tool in characterization between the ER+ subtypes, Luminal A, and Luminal B. Conclusions: We show that miR-190b targets RFWD3 in ER+ breast cancers leading to lower RFWD3 protein expression. Low levels of RFWD3 are associated with better outcomes in patients with Luminal A breast cancer but not in patients with Luminal B breast cancer. These findings provide novel insights into miR-190b role in breast cancer and that its clinical relevance is subtype specific

The Chromatin Scaffold Protein SAFB1 Renders Chromatin Permissive for DNA Damage Signaling

Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling