GATA3 mRNA expression, but not mutation, associates with longer progression-free survival in ER-positive breast cancer patients treated with first-line tamoxifen for recurrent disease

In breast cancer, GATA3 mutations have been associated with a favorable prognosis and the response to neoadjuvant aromatase inhibitor treatment. Therefore, we investigated whether GATA3 mutations predict the outcome of tamoxifen treatment in the advanced setting. In a retrospective study consisting of 235 hormone-naive patients with ER-positive breast cancer who received tamoxifen as first-line treatment for recurrent disease, GATA3 mutations (in 14.0% of patients) did not significantly associate with either the overall response rate (ORR) or with the length of progression-free survival (PFS) after the start of tamoxifen therapy. Interestingly, among 148 patients for whom both mutation and mRNA expression data were available, GATA3 mutations associated with an increased expression of GATA3. However, only 23.7% of GATA3 high tumors had a mutation. Evaluation of the clinical significance of GATA3 mRNA revealed that it was associated with prolonged PFS, but not with the ORR, also in multivariate analysis. Thus, GATA3 mRNA expression, but not GATA3 mutation, is an independent predictor of prolonged PFS in ER-positive breast cancer patients who received first-line tamoxifen for recurrent disease. Besides GATA3 mutation, other mechanisms must exist that underlie increased GATA3 levels

Understanding drugs in breast cancer through drug sensitivity screening

With substantial numbers of breast tumors showing or acquiring treatment resistance, it is of utmost importance to develop new agents for the treatment of the disease, to know their effectiveness against breast cancer and to understand their relationships with other drugs to best assign the right drug to the right patient. To achieve this goal drug screenings on breast cancer cell lines are a promising approach. In this study a large-scale drug screening of 37 compounds was performed on a panel of 42 breast cancer cell lines representing the main breast cancer subtypes. Clustering, correlation and pathway analyses were used for data analysis. We found that compounds with a related mechanism of action had correlated IC50 values and thus grouped together when the cell lines were hierarchically clustered based on IC50 values. In total we found six clusters of drugs of which five consisted of drugs with related mode of action and one cluster with two drugs not previously connected. In total, 25 correlated and four anti-correlated drug sensitivities were revealed of which only one drug, Sirolimus, showed significantly lower IC50 values in the luminal/ERBB2 breast cancer subtype. We found expected interactions but also discovered new relationships between

Recurrent HOXB13 mutations in the Dutch population do not associate with increased breast cancer risk

The HOXB13 p.G84E mutation has been firmly established as a prostate cancer susceptibility allele. Although HOXB13 also plays a role in breast tumor progression, the association of HOXB13 p.G84E with breast cancer risk is less evident. Therefore, we comprehensively interrogated the entire HOXB13 coding sequence for mutations in 1,250 non-BRCA1/2 familial breast cancer cases and 800 controls. We identified two predicted deleterious missense mutations, p.G84E and p.R217C, that were recurrent among breast cancer cases and further evaluated their association with breast cancer risk in a larger study. Taken together, 4,520 familial non-BRCA1/2 breast cancer cases and 3,127 controls were genotyped including the cases and controls of the whole gene screen. The concordance rate for the genotyping assays compared with Sanger sequencing was 100%. The prostate cancer risk allele p.G84E was identified in 18 (0.56%) of 3,187 cases and 16 (0.70%) of 2,300 controls (OR = 0.81, 95% CI = 0.41-1.59, P = 0.54). Additionally, p.R217C was identified in 10 (0.31%) of 3,208 cases and 2 (0.087%) of 2,288 controls (OR = 3.57, 95% CI = 0.76-33.57, P = 0.14). These results imply that none of the recurrent HOXB13 mutations in the Dutch population are associated with breast cancer risk, although it may be worthwhile to evaluate p.R217C in a larger study

Expression and Localization of Ferritin-Heavy Chain Predicts Recurrence for Breast Cancer Patients with a <i>BRCA1/2</i> Mutation

The ferritin-heavy chain (FTH1) is the catalytic subunit of the ferroxidase ferritin, which prevents oxidative DNA damage via intracellular iron storage. FTH1 was shown to be a prognostic marker for triple-negative breast cancer (BC) patients and associated with an enrichment of CD8+ effector T cells. However, whether the expression and localization of FTH1 are also associated with clinical outcome in other BC subtypes is unknown. Here, we investigated the association of FTH1 with time to survival in BCs from 222 BRCA1/2 mutation carriers by immunohistochemistry on tissue microarrays. In addition, for 51 of these patients, the association between FTH1 and specific subsets of T cells was evaluated on whole slides using automatic scoring algorithms. We revealed that nuclear FTH1 (nFTH1) expression, in multivariable analyses, was associated with a shorter disease-free (HR = 2.71, 95% CI = 1.49–4.92, p = 0.001) and metastasis-free survival (HR = 3.54, 95% CI = 1.45–8.66, p = 0.006) in patients carrying a BRCA1/2 mutation. However, we found no relation between cytoplasmic FTH1 expression and survival of BRCA1/2 mutation carriers. Moreover, we did not detect an association between FTH1 expression and the amount of CD45+ (p = 0.13), CD8+ (p = 0.18), CD4+ (p = 0.20) or FOXP3+ cells (p = 0.17). Consequently, the mechanism underlying the worse recurrence-free survival of nFTH1 expression in BRCA1/2 mutation carriers needs further investigation.</p

MicroRNAs as possible indicators of drug sensitivity in breast cancer cell lines

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally. In this way they might influence whether a cell is sensitive or resistant to a certain drug. So far, only a limited number of relatively small scale studies comprising few cell lines and/or drugs have been performed. To obtain a broader view on miRNAs and their association with drug response, we investigated the expression levels of 411 miRNAs in relation to drug sensitivity in 36 breast cancer cell lines. For this purpose IC50 values of a drug screen involving 34 drugs were associated with miRNA expression data of the same breast cancer cell lines. Since molecular subtype of the breast cancer cell lines is considered a confounding factor in drug association studies, multivariate analysis taking subtype into account was performed on significant miRNA-drug associations which retained 13 associations. These associations consisted of 11 different miRNAs and eight different drugs (among which Paclitaxel, Docetaxel and Veliparib). The taxanes, Paclitaxel and Docetaxel, were the only drugs having miRNAs in common: hsa-miR-187-5p and hsa-miR-106a-3p indicative of drug resistance while Paclitaxel sensitivity alone associated with hsa-miR-556-5p. Tivantinib was associated with hsalet-7d-5p and hsa-miR-18a-5p for sensitivity and hsa-miR-637 for resistance. Drug sensitivity was associated with hsa-let-7a-5p for Bortezomib, hsa-miR-135a-3p for JNJ-707 and hsa-miR-185-3p for Panobinostat. Drug resistance was associated with hsa-miR-182-5p for Veliparib and hsa-miR-629-5p for Tipifarnib. Pathway analysis for significant miRNAs was performed to reveal biological roles, aiding to find a potential mechanistic link for the observed associations with drug response. By doing so hsa-miR-187-5p was linked to the cell cycle G2-M checkpoint in line with this checkpoint being the target of taxanes. In conclusion, our study shows that miRNAs could potentially serve as biomarkers for intrinsic drug resistance and that pathway analyses can provide additional information in this contex

MiRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs

Introduction: Breast cancer is a genetically and phenotypically complex disease. To understand the role of miRNAs in this molecular complexity, we performed miRNA expression analysis in a cohort of molecularly well-characterized human breast cancer cell lines to identify miRNAs associated with the most common molecular subtypes and the most frequent genetic aberrations. Methods: Using a microarray carrying LNA™ modified oligonucleotide capture probes), expression levels of 725 human miRNAs were measured in 51 breast cancer cell lines. Differential miRNA expression was explored by unsupervised cluster analysis and was then associated with the molecular subtypes and genetic aberrations commonly present in breast cancer. Results: Unsupervised cluster analysis using the most variably expressed miRNAs divided the 51 breast cancer cell lines into a major and a minor cluster predominantly mirroring the luminal and basal intrinsic subdivision of breast cancer cell lines. One hundred and thirteen miRNAs were differentially expressed between these two main clusters. Forty miRNAs were differentially expressed between basal-like and normal-like/claudin-low cell lines. Within the luminal-group, 39 miRNAs were associated with ERBB2 overexpression and 24 with E-cadherin gene mutations, which are frequent in this subtype of breast cancer cell lines. In contrast, 31 miRNAs were associated with E-cadherin promoter hypermethylation, which, contrary to E-cadherin mutation, is exclusively observed in breast cancer cell lines that are not of luminal origin. Thirty miRNAs were associated with p16INK4 status while only a fe

Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids?

Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings. We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ~70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance. We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter

ATG24 represses autophagy and differentiation and is essential for homeostasy of the flagellar pocket in trypanosoma brucei

We have previously identified homologs for nearly half of the approximately 30 known yeast Atg's in the genome database of the human sleeping sickness parasite Trypanosoma brucei. So far, only a few of these homologs have their role in autophagy experimentally confirmed. Among the candidates was the ortholog of Atg24 that is involved in pexophagy in yeast. In T. brucei, the peroxisome-like organelles named glycosomes harbor core metabolic processes, especially glycolysis. In the autotrophic yeast, autophagy is essential for adaptation to different nutritional environments by participating in the renewal of the peroxisome population. We hypothesized that autophagic turnover of the parasite's glycosomes plays a role in differentiation during its life cycle, which demands adaptation to different host environments and associated dramatic changes in nutritional conditions. We therefore characterized T. brucei ATG24, the T. brucei ortholog of yeast Atg24 and mammalian SNX4, and found it to have a regulatory role in autophagy and differentiation as well as endocytic trafficking. ATG24 partially localized on endocytic membranes where it was recruited via PI3-kinase III/VPS34. ATG24 silencing severely impaired receptor-mediated endocytosis of transferrin, but not adsorptive uptake of a lectin, and caused a major enlargement of the flagellar pocket. ATG24 silencing approximately doubled the number of autophagosomes, suggesting a role in repressing autophagy, and strongly accelerated differentiation, in accordance with a role of autophagy in parasite differentiation. Overexpression of the two isoforms of T. brucei ATG8 fused to GFP slowed down differentiation, possibly by a dominant-negative effect. This was overcome by ATG24 depletion, further supporting its regulatory role

Germline HOXB13 mutations p.G84E and p.R217C do not confer an increased breast cancer risk

In breast cancer, high levels of homeobox protein Hox-B13 (HOXB13) have been associated with disease progression of ER-positive breast cancer patients and resistance to tamoxifen treatment. Since HOXB13 p.G84E is a prostate cancer risk allele, we evaluated the association between HOXB13 germline mutations and breast cancer risk in a previous study consisting of 3,270 familial non-BRCA1/2 breast cancer cases and 2,327 controls from the Netherlands. Although both recurrent HOXB13 mutations p.G84E and p.R217C were not associated with breast cancer risk, the risk estimation for p.R217C was not very precise. To provide more conclusive evidence regarding the role of HOXB13 in breast cancer susceptibility, we here evaluated the association between HOXB13 mutations and increased breast cancer risk within 81 studies of the international Breast Cancer Association Consortium containing 68,521 invasive breast cancer patients and 54,865 controls. Both HOXB13 p.G84E and p.R217C did not associate with the development of breast cancer in European women, neither in the overall analysis (OR = 1.035, 95% CI = 0.859-1.246, P = 0.718 and OR = 0.798, 95% CI = 0.482-1.322, P = 0.381 respectively), nor in specific high-risk subgroups or breast cancer subtypes. Thus, although involved in breast cancer progression, HOXB13 is not a material breast cancer susceptibility gene