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Liver X receptors (LXRs) are nuclear transcription factors important in the regulation of cholesterol transport, and glucose and fatty acid metabolism. The antiproliferative role of LXRs has been studied in a variety of malignancies and may represent a therapeutic opportunity in cancers lacking targeted therapies, such as triple-negative breast cancer. In this study, we investigated the impact of LXR agonists alone and in combination with carboplatin in preclinical models of breast cancer. In vitro experiments revealed a dose-dependent decrease in tumor cell proliferation in estrogen receptor-positive breast cancer cells, whereas LXR activation in vivo resulted in an increased growth inhibitory effect in a basal-like breast cancer model (in combination with carboplatin). Functional proteomic analysis identified differences in protein expression between responding and nonresponding models related to Akt activity, cell-cycle progression, and DNA repair. Furthermore, pathway analysis suggested that the LXR agonist in combination with carboplatin inhibits the activity of targets of E2F transcription factors and affects cholesterol homeostasis in basal-like breast cancer., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Alterations in glycan structures are early signs of malignancy and have recently been proposed to be in part a driving force behind malignant transformation. Here, we explore whether differences in expression of genes related to the process of glycosylation exist between breast carcinoma subtypes - and look for their association to clinical parameters. Five expression datasets of 454 invasive breast carcinomas, 31 ductal carcinomas in situ (DCIS), and 79 non-malignant breast tissue samples were analysed. Results were validated in 1960 breast carcinomas. 419 genes encoding glycosylation-related proteins were selected. The DCIS samples appeared expression-wise similar to carcinomas, showing altered gene expression related to glycosaminoglycans (GAGs) and N-glycans when compared to non-malignant samples. In-situ lesions with different aggressiveness potentials demonstrated changes in glycosaminoglycan sulfation and adhesion proteins. Subtype-specific expression patterns revealed down-regulation of genes encoding glycan-binding proteins in the luminal A and B subtypes. Clustering basal-like samples using a consensus list of genes differentially expressed across discovery datasets produced two clusters with significantly differing prognosis in the validation dataset. Finally, our analyses suggest that glycolipids may play an important role in carcinogenesis of breast tumors - as demonstrated by association of B3GNT5 and UGCG genes to patient survival. In conclusion, most glycan-specific changes occur early in the carcinogenic process. We have identified glycan-related alterations specific to breast cancer subtypes including a prognostic signature for two basal-like subgroups. Future research in this area may potentially lead to markers for better prognostication and treatment stratification of breast cancer patients., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

For a panel of cancer related proteins, the aim was to shed light on which molecular level the expression of each protein was mainly regulated in breast tumors, and to investigate whether differences in regulation were reflected in different molecular subtypes. DNA, mRNA and protein lysates from 251 breast tumor specimens were analyzed using appropriate microarray technologies. Data from all three levels were available for 52 proteins selected for their known involvement in cancer, primarily through the PI3K/Akt pathway. For every protein, in cis Spearman rank correlations between the three molecular levels were calculated across all samples and within each intrinsic gene expression subtype, enabling 63 comparisons altogether due to multiple gene probes matching to single proteins. Subtype-specific relationships between the three molecular levels were studied by calculating the variance of subtype-specific correlation and differences between overall and average subtype-specific correlation. The findings were validated in an external dataset comprising 703 breast tumor specimens. The proteins were sorted into four groups based on the calculated rank correlation values between the three molecular levels. Group A consisted of eight proteins with significant correlation between DNA copy number levels and mRNA expression, and between mRNA expression and protein expression (Bonferroni adjusted p < 0.05). Group B consisted of 14 proteins with significant correlation between mRNA expression and protein expression. Group C consisted of 15 proteins with significant correlation between copy number levels and mRNA expression. For the remaining 25 proteins (group D), no significant correlations was observed. Stratification of tumors according to intrinsic subtype enabled identification of positive correlations between copy number levels, mRNA and protein expression that were undetectable when considering the entire sample set. Protein pairings that either demonstrated high variance in correlation values between subtypes, or between subtypes and the total dataset were studied in particular. The protein expression of cleaved caspase 7 was most highly expressed, and correlated highest to CASP7 gene expression within the basal-like subtype, accompanied by the lowest amounts of hsa-miR-29c. Luminal A-like subtype demonstrated highest amounts of hsa-miR-29c (a miRNA with a putative target sequence in CASP7 mRNA), low expression of cleaved caspase 7 and low correlation to CASP7 gene expression. Such pattern might be an indication of hsa-miR-29c miRNA functioning as a repressor of translation of CASP7 within the luminal-A subtype. Across the entire cohort no correlation was found between CCNB1 copy number and gene expression. However, within most gene intrinsic subtypes, mRNA and protein expression of cyclin B1 was found positively correlated to copy number data, suggesting that copy number can affect the overall expression of this protein. Aberrations of cyclin B1 copy number also identified patients with reduced overall survival within each subtype. Based on correlation between the three molecular levels, genes and their products could be sorted into four groups for which the expression was likely to be regulated at different molecular levels. Further stratification suggested subtype-specific regulation that was not evident across the entire sample set., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Antiangiogenic therapy with bevacizumab has shown varying results in breast cancer clinical trials. Identifying robust biomarkers for selecting patients who may benefit from such treatment and for monitoring response is important for the future use of bevacizumab. Two established xenograft models representing basal-like and luminal-like breast cancer were used to study bevacizumab treatment response on the metabolic and gene expression levels. Tumor samples were obtained from mice treated with bevacizumab, doxorubicin or a combination of the two drugs, and high resolution magic angle spinning magnetic resonance spectroscopy and gene expression microarray analysis was performed. Combination treatment with bevacizumab showed the strongest growth inhibiting effect in basal-like tumors, and this was reflected by a significant change in the metabolomic and transcriptomic profiles. In the luminal-like xenografts, addition of bevacizumab did not improve the effect of doxorubicin. On the global transcriptomic level, the largest gene expression changes were observed for the most efficient treatment in both models. Glycerophosphocholine showed opposite response in the treated xenografts compared with untreated controls; lower in basal-like and higher in luminal-like tumors. Comparing combination therapy with doxorubicin monotherapy in basal-like xenografts, 14 genes showed significant differential expression, including very low density lipoprotein receptor (VLDLR) and hemoglobin, theta 1 (HBQ1). Bevacizumab-treated tumors were associated with a more hypoxic phenotype, while no evidence was found for associations between bevacizumab treatment and vascular invasion or tumor grade. This study underlines the importance of characterizing biological differences between subtypes of breast cancer to identify personalized biomarkers for improved patient stratification and evaluation of response to therapy., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Time to freezing tumor tissue for RNA expression analysis will always vary to some extent. To evaluate the effect of ischemia time, tumor tissue from ten breast cancer patients was collected and aliquots of tissue were snap frozen at different time points after surgery (0, 0.5, 1, 3 and 6 h). Using miRNA and mRNA expression microarrays and statistical analysis, 56 miRNAs and 1788 mRNAs were found to be significantly altered with ischemia time up to six hours. Several of the 56 miRNAs have been reported to play a role in cancer, such as hsa-miR-663 and hsa-miR-125a-3p. Known stress response genes such as GADD45B, JUND and FOSB were among the mRNAs most significantly affected by time to freezing. A novel statistical method for identification of consistently correlated miRNA-mRNA pairs and miRNA-associated biological processes in time course data is presented. Application of this method revealed that several miRNAs, including hsa-miR-1228, hsa-miR-1225-5p and hsa-miR-574-5p, were associated through their correlation to mRNAs to biological processes such as "response to stimulus" and "stress response". These miRNAs also showed enrichment of predicted targets among either their positively or negatively correlated mRNAs. The induced miRNAs may play both direct and indirect roles in biological responses. Caution should be taken when the miRNAs and mRNAs reported to be affected by ischemia time are included in a prognostic or predictive signature., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Ductal carcinoma in situ (DCIS) is a non-invasive form of breast cancer where cells restricted to the ducts exhibit an atypical phenotype. Some DCIS lesions are believed to rapidly transit to invasive ductal carcinomas (IDCs), while others remain unchanged. Existing classification systems for DCIS fail to identify those lesions that transit to IDC. We studied gene expression patterns of 31 pure DCIS, 36 pure invasive cancers and 42 cases of mixed diagnosis (invasive cancer with an in situ component) using Agilent Whole Human Genome Oligo Microarrays 44k. Six normal breast tissue samples were also included as controls. qRT-PCR was used for validation. All DCIS and invasive samples could be classified into the "intrinsic" molecular subtypes defined for invasive breast cancer. Hierarchical clustering establishes that samples group by intrinsic subtype, and not by diagnosis. We observed heterogeneity in the transcriptomes among DCIS of high histological grade and identified a distinct subgroup containing seven of the 31 DCIS samples with gene expression characteristics more similar to advanced tumours. A set of genes independent of grade, ER-status and HER2-status was identified by logistic regression that univariately classified a sample as belonging to this distinct DCIS subgroup. qRT-PCR of single markers clearly separated this DCIS subgroup from the other DCIS, and contains samples from several histopathological and intrinsic molecular subtypes. The genes that differentiate between these two types of DCIS suggest several processes related to the re-organisation of the microenvironment. This raises interesting possibilities for identification of DCIS lesions both with and without invasive characteristics, which potentially could be used in clinical assessment of a woman's risk of progression, and lead to improved management that would avoid the current over- and under-treatment of patients., ((c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Triple-negative breast cancers (TNBC), characterized by absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor 2 (HER2), are typically associated with poor prognosis, due to aggressive tumor phenotype(s), only partial response to chemotherapy and present lack of clinically established targeted therapies. Advances in the design of individualized strategies for treatment of TNBC patients require further elucidation, by combined 'omics' approaches, of the molecular mechanisms underlying TNBC phenotypic heterogeneity, and the still poorly understood association of TNBC with BRCA1 mutations. An overview is here presented on TNBC profiling in terms of expression signatures, within the functional genomic breast tumor classification, and ongoing efforts toward identification of new therapy targets and bioimaging markers. Due to the complexity of aberrant molecular patterns involved in expression, pathological progression and biological/clinical heterogeneity, the search for novel TNBC biomarkers and therapy targets requires collection of multi-dimensional data sets, use of robust multivariate data analysis techniques and development of innovative systems biology approaches., ((c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Glycosylation is the stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process have been associated with malignant transformation. Simultaneous analysis of the expression of all glycan-related genes clearly gives the advantage of enabling a comprehensive view of the genetic background of the glycobiological changes in cancer cells. Studies focusing on the expression of the whole glycome have now become possible, which prompted us to review the present knowledge on glycosylation in relation to breast cancer diagnosis and progression, in the light of available expression data from tumors and breast tissue of healthy individuals. We used various data resources to select a set of 419 functionally relevant genes involved in synthesis, degradation and binding of N-linked and O-linked glycans, Lewis antigens, glycosaminoglycans (chondroitin, heparin and keratan sulfate in addition to hyaluronan) and glycosphingolipids. Such glycans are involved in a number of processes relevant to carcinogenesis, including regulation of growth factors/growth factor receptors, cell-cell adhesion and motility as well as immune system modulation. Expression analysis of these glycan-related genes revealed that mRNA levels for many of them differ significantly between normal and malignant breast tissue. An associative analysis of these genes in the context of current knowledge of their function in protein glycosylation and connection(s) to cancer indicated that synthesis, degradation and adhesion mediated by glycans may be altered drastically in mammary carcinomas. Although further analysis is needed to assess how changes in mRNA levels of glycan genes influence a cell's glycome and the precise role that such altered glycan structures play in the pathogenesis of the disease, lessons drawn from this study may help in determining directions for future research in the rapidly-developing field of glycobiology., (Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The number of relevant and well-characterized cell lines and xenograft models for studying human breast cancer are few, and may represent a limitation for this field of research. With the aim of developing new breast cancer model systems for in vivo studies of hormone dependent and independent tumor growth, progression and invasion, and for in vivo experimental therapy studies, we collected primary mammary tumor specimens from patients, and implanted them in immunodeficient mice. Primary tumor tissue from 29 patients with breast cancer was implanted subcutaneously with matrigel in SCID mice, in the presence of continuous release of estradiol. The tumors were transferred into new animals when reaching a diameter of 15mm and engrafted tumors were harvested for morphological and molecular characterization from passage six. Further, gene expression profiling was performed using Agilent Human Whole Genome Oligo Microarrays, as well as DNA copy number analysis using Agilent Human Genome CGH 244K Microarrays. Of the 30 primary tumors implanted into mice (including two implants from the same patient), two gave rise to viable tumors beyond passage ten. One showed high expression levels of estrogen receptor-alpha protein (ER) while the other was negative. Histopathological evaluation of xenograft tumors was carried out at passage 10-12; both xenografts maintained the morphological characteristics of the original tumors (classified as invasive grade III ductal carcinomas). The genomic profile of the ER-positive xenograft tumor resembled the profile of the primary tumor, while the profile obtained from the ER-negative parental tumor was different from the xenograft. However, the ER-negative parental tumor and xenograft clustered on the same branch using unsupervised hierarchical clustering analysis on RNA microarray expression data of "intrinsic genes". A significant variation was observed in the expression of extracellular matrix (ECM)-related genes, which were found downregulated in the engrafted tumors compared to the primary tumor. By IHC and qRT-PCR we found that the downregulation of stroma-related genes was compensated by the overexpression of such molecules by the mouse host tissue. The two established breast cancer xenograft models showed different histopathological characteristics and profound diversity in gene expression patterns that in part can be associated to their ER status and here described as basal-like and luminal-like phenotype, respectively. These two new breast cancer xenografts represent useful preclinical tools for developing and testing of new therapies and improving our knowledge on breast cancer biology.

In breast cancer, previous studies have suggested that somatic TP53 mutations are likely to be an early event. However, there are controversies regarding the cellular origin and linear course of breast cancer. The purpose of this study was to investigate tumor evolution in breast cancer by analyzing TP53 mutation status in tumors from various stages of the disease. The entire coding sequence of TP53 was sequenced in a cohort of pure ductal carcinoma in situ (DCIS), pure invasive cancer (≤15mm) and mixed lesions (i.e. invasive cancer with an in situ component). Of 118 tumor samples, 19 were found to harbor a TP53 mutation; 5 (15.6%) of the pure DCIS, 4 (10.5%) of the pure invasive cancers and 10 (20.8%) of the mixed lesions. In the mixed lesions, both the invasive and the DCIS components showed the same mutation in all 5 cases where the two components were successfully microdissected. Presence of the same mutation in both DCIS and invasive components from the same tumor indicates same cellular origin. The role of mutant TP53 in the progression of breast cancer is less clear and may vary between subtypes.

Expression profiles of primary breast tumors were investigated in relation to disseminated tumor cells (DTCs) in bone marrow (BM) in order to increase our understanding of the dissemination process. Tumors were classified into five pre-defined molecular subtypes, and presence of DTC identified (at median 85 months follow-up) a subgroup of luminal A patients with particular poor outcome (p=0.008). This was not apparent for other tumor subtypes. Gene expression profiles associated with DTC and with systemic relapse for luminal A patients were identified. This study suggests that DTC in BM differentially distinguishes clinical outcome in patients with luminal A type tumors and that DTC-associated gene expression analysis may identify genes of potential importance in tumor dissemination.