Inhibition of PTP1B disrupts cell-cell adhesion and induces anoikis in breast epithelial cells.

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 FilesProtein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell-cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype.Landspitali University Hospital Science Fund University of Iceland Research Fund Icelandic Science and Technology Policy Council Research Fund Icelandic Science and Technology Policy - Grant of Excellence Gongum sama

Metabolic re-wiring of isogenic breast epithelial cell lines following epithelial to mesenchymal transition.

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 FilesEpithelial to mesenchymal transition (EMT) has implications in tumor progression and metastasis. Metabolic alterations have been described in cancer development but studies focused on the metabolic re-wiring that takes place during EMT are still limited. We performed metabolomics profiling of a breast epithelial cell line and its EMT derived mesenchymal phenotype to create genome-scale metabolic models descriptive of both cell lines. Glycolysis and OXPHOS were higher in the epithelial phenotype while amino acid anaplerosis and fatty acid oxidation fueled the mesenchymal phenotype. Through comparative bioinformatics analysis, PPAR-γ1, PPAR- γ2 and AP-1 were found to be the most influential transcription factors associated with metabolic re-wiring. In silico gene essentiality analysis predicts that the LAT1 neutral amino acid transporter is essential for mesenchymal cell survival. Our results define metabolic traits that distinguish an EMT derived mesenchymal cell line from its epithelial progenitor and may have implications in cancer progression and metastasis. Furthermore, the tools presented here can aid in identifying critical metabolic nodes that may serve as therapeutic targets aiming to prevent EMT and inhibit metastatic dissemination.Icelandic Research Counci

Endothelial induced EMT in breast epithelial cells with stem cell properties.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.Landspitali University Hospital University of Iceland Science and Technology Policy Council European Science Foundatio

HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR.

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 Files. This article is open access.The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal breast epithelium, what effects they have on cellular differentiation and how they participate in neoplastic transformation. D492 is a breast epithelial cell line with stem cell properties that can undergo epithelial to mesenchyme transition (EMT), generate luminal- and myoepithelial cells and form complex branching structures in three-dimensional (3D) culture. Here, we show that overexpression of HER2 in D492 (D492(HER2)) resulted in EMT, loss of contact growth inhibition and increased oncogenic potential in vivo. HER2 overexpression, furthermore, inhibited endogenous EGFR expression. Re-introducing EGFR in D492(HER2) (D492(HER2/EGFR)) partially reversed the mesenchymal state of the cells, as an epithelial phenotype reappeared both in 3D cultures and in vivo. The D492(HER2/EGFR) xenografts grow slower than the D492(HER2) tumors, while overexpression of EGFR alone (D492(EGFR)) was not oncogenic in vivo. Consistent with the EGFR-mediated epithelial phenotype, overexpression of EGFR drove the cells toward a myoepithelial phenotype in 3D culture. The effect of two clinically approved anti-HER2 and EGFR therapies, trastuzumab and cetuximab, was tested alone and in combination on D492(HER2) xenografts. While trastuzumab had a growth inhibitory effect compared with untreated control, the effect of cetuximab was limited. When administered in combination, the growth inhibitory effect of trastuzumab was less pronounced. Collectively, our data indicate that in HER2-overexpressing D492 cells, EGFR can behave as a tumor suppressor, by pushing the cells towards epithelial differentiation.Landspitali University Hospital Science Fund, University of Iceland Research Fund, Science and Technology Policy Council Research Fund and Grant of Excellence, ‘Göngum saman’, a supporting group for breast cancer research in Iceland

Trichomonas vaginalis is Rare Among Women in Iceland

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 FilesLandspitali University Hospital Orebro County Council Research Committee Foundation for Medical Research at Orebro University Hospital, Orebro, Swede

A DNA methylation-based definition of biologically distinct breast cancer subtypes

In cancer, epigenetic states are deregulated and thought to be of significance in cancer development and progression. We explored DNA methylation-based signatures in association with breast cancer subtypes to assess their impact on clinical presentation and patient prognosis. DNA methylation was analyzed using Infinium 450K arrays in 40 tumors and 17 normal breast samples, together with DNA copy number changes and subtype-specific markers by tissue microarrays. The identified methylation signatures were validated against a cohort of 212 tumors annotated for breast cancer subtypes by the PAM50 method (The Cancer Genome Atlas). Selected markers were pyrosequenced in an independent validation cohort of 310 tumors and analyzed with respect to survival, clinical stage and grade. The results demonstrate that DNA methylation patterns linked to the luminal-B subtype are characterized by CpG island promoter methylation events. In contrast, a large fraction of basal-like tumors are characterized by hypomethylation events occurring within the gene body. Based on these hallmark signatures, we defined two DNA methylation-based subtypes, Epi-LumB and Epi-Basal, and show that they are associated with unfavorable clinical parameters and reduced survival. Our data show that distinct mechanisms leading to changes in CpG methylation states are operative in different breast cancer subtypes. Importantly, we show that a few selected proxy markers can be used to detect the distinct DNA methylation-based subtypes thereby providing valuable information on disease prognosis

Hsa-miR-21-3p associates with breast cancer patient survival and targets genes in tumor suppressive pathways.

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 DownloadBreast cancer is the cancer most often diagnosed in women. MicroRNAs (MIRs) are short RNA molecules that bind mRNA resulting in their downregulation. MIR21 has been shown to be an oncomiR in most cancer types, including breast cancer. Most of the effects of miR-21 have been attributed to hsa-miR-21-5p that is transcribed from the leading strand of MIR21, but hsa-miR-21-3p (miR-21-3p), transcribed from the lagging strand, is much less studied. The aim of the study is to analyze whether expression of miR-21-3p is prognostic for breast cancer. MiR-21-3p association with survival, clinical and pathological characteristics was analyzed in a large breast cancer cohort and validated in three separate cohorts, including TCGA and METABRIC. Analytical tools were also used to infer miR-21-3p function and to identify potential target genes and functional pathways. The results showed that in the exploration cohort, high miR-21-3p levels associated with shorter survival and lymph node positivity. In the three validation cohorts, high miR-21-3p levels associated with pathological characteristics that predict worse prognosis. Specifically, in the largest validation cohort, METABRIC (n = 1174), high miR-21-3p levels associated with large tumors, a high grade, lymph node and HER2 positivity, and shorter breast-cancer-specific survival (HR = 1.38, CI 1.13-1.68). This association remained significant after adjusting for confounding factors. The genes with expression levels that correlated with miR-21-3p were enriched in particular pathways, including the epithelial-to-mesenchymal transition and proliferation. Among the most significantly downregulated targets were MAT2A and the tumor suppressive genes STARD13 and ZNF132. The results from this study emphasize that both 3p- and 5p-arms from a MIR warrant independent study. The data show that miR-21-3p overexpression in breast tumors is a marker of worse breast cancer progression and it affects genes in pathways that drive breast cancer by down-regulating tumor suppressor genes. The results suggest miR-21-3p as a potential biomarker.This research was funded by grants to IR, RBB, BAA, OTJ and AdAr from the Icelandic Centre for Research (grant number 152530-051, www.rannis.is), The Scientific Fund of Landspitali – The National University Hospital in Iceland (grant numbers A-2016-033 and A-2019-042, www.landspitali.is), The Scientific Fund of The Icelandic Cancer Society (year 2017, www.krabb.is/english/), Gongum saman (https://gongumsaman.is/; years 2013 and 2017) and a grant to ArAm and IR from Gongum saman in 2018. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript

BRCA1 Promoter Methylation Status in 1031 Primary Breast Cancers Predicts Favorable Outcomes Following Chemotherapy.

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 DownloadBackground: Breast Cancer 1 gene (BRCA1) is known to be inactivated in breast tumors by promoter methylation. Tumor cells in patients carrying a germline mutation in BRCA1 are sensitive to cytotoxic drugs that cause DNA double strand breaks. However, very little is known on whether patients with BRCA1 promoter methylated tumors are similarly sensitive to cytotoxic drugs. In this study, we address this by making use of extensive follow-up data on patients treated with cyclophosphamide, methotrexate, and fluorouracil in Iceland between 1976 and 2007. Methods: We analyzed BRCA1 promoter methylation by pyrosequencing DNA from tumor samples from 1031 patients with primary breast cancer. Of those, 965 were sporadic cases, 61 were BRCA2, and five were BRCA1 germline mutation carriers. All cases were examined with respect to clinicopathological parameters and breast cancer-specific survival in patients treated with cytotoxic drugs. Information on chemotherapy treatment in noncarriers was available for 26 BRCA1 methylated tumors and 857 unmethylated tumors. Results: BRCA1 was promoter methylated in 29 sporadic tumors or in 3.0% of cases (29 of 965), whereas none of the tumors derived from BRCA germline mutation carriers were promoter methylated. Important to note, patients with BRCA1 promoter methylation receiving chemotherapeutic drug treatment show highly improved breast cancer-specific survival compared with unmethylated controls (hazard ratio = 0.10, 95% confidence interval = 0.01 to 0.75, two-sided P = .02). Conclusions: BRCA1 promoter methylation is predictive of improved disease outcome in patients receiving cyclophosphamide, methotrexate, and fluorouracil drug treatment. Our results support the use of markers indicative of "BRCAness" in sporadic breast cancers to identify patients that are likely to benefit from the use of DNA-damaging agents.Icelandic Research Fund Gongum sama

Expression of ncRNAs on the DLK1-DIO3 Locus Is Associated With Basal and Mesenchymal Phenotype in Breast Epithelial Progenitor Cells.

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 DownloadEpithelial-to-mesenchymal transition (EMT) and its reversed process mesenchymal-to-epithelial transition (MET) play a critical role in epithelial plasticity during development and cancer progression. Among important regulators of these cellular processes are non-coding RNAs (ncRNAs). The imprinted DLK1-DIO3 locus, containing numerous maternally expressed ncRNAs including the lncRNA maternally expressed gene 3 (MEG3) and a cluster of over 50 miRNAs, has been shown to be a modulator of stemness in embryonic stem cells and in cancer progression, potentially through the tumor suppressor role of MEG3. In this study we analyzed the expression pattern and functional role of ncRNAs from the DLK1-DIO3 locus in epithelial plasticity of the breast. We studied their expression in various cell types of breast tissue and revisit the role of the locus in EMT/MET using a breast epithelial progenitor cell line (D492) and its isogenic mesenchymal derivative (D492M). Marked upregulation of ncRNAs from the DLK1-DIO3 locus was seen after EMT induction in two cell line models of EMT. In addition, the expression of MEG3 and the maternally expressed ncRNAs was higher in stromal cells compared to epithelial cell types in primary breast tissue. We also show that expression of MEG3 is concomitant with the expression of the ncRNAs from the DLK1-DIO3 locus and its expression is therefore likely indicative of activation of all ncRNAs at the locus. MEG3 expression is correlated with stromal markers in normal tissue and breast cancer tissue and negatively correlated with the survival of breast cancer patients in two different cohorts. Overexpression of MEG3 using CRISPR activation in a breast epithelial cell line induced partial EMT and enriched for a basal-like phenotype. Conversely, knock down of MEG3 using CRISPR inhibition in a mesenchymal cell line reduced the mesenchymal and basal-like phenotype of the cell line. In summary our study shows that maternally expressed ncRNAs are markers of EMT and suggests that MEG3 is a novel regulator of EMT/MET in breast tissue. Nevertheless, further studies are needed to fully dissect the molecular pathways influenced by non-coding RNAs at the DLK1-DIO3 locus in breast tissue.Landspitali University Hospital Science Fund Icelandic Science and Technology Policy Council Research Fund Icelandic Science and Technology Policy - Grant of Excellence "Visindasjogur Krabbameinsfelagsins" (Icelandic Cancer Society Science Fund) 2017 University of Iceland Research Fund 'Gongum saman', a supporting group for breast cancer research in Icelan

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, CK8, CK18, EGFR, MUC1, and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥ 40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response