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

Vps34/PI3KC3 deletion in kidney proximal tubules impairs apical trafficking and blocks autophagic flux, causing a Fanconi-like syndrome and renal insufficiency

Kidney proximal tubular cells (PTCs) are highly specialized for ultrafiltrate reabsorption and serve as paradigm of apical epithelial differentiation. Vps34/PI3-kinase type III (PI3KC3) regulates endosomal dynamics, macroautophagy and lysosomal function. However, its in vivo role in PTCs has not been evaluated. Conditional deletion of Vps34/PI3KC3 in PTCs by Pax8-Cre resulted in early (P7) PTC dysfunction, manifested by Fanconi-like syndrome, followed by kidney failure (P14) and death. By confocal microscopy, Vps34∆/∆ PTCs showed preserved apico-basal specification (brush border, NHERF-1 versus Na+/K+-ATPase, ankyrin-G) but basal redistribution of late-endosomes/lysosomes (LAMP-1) and mis-localization to lysosomes of apical recycling endocytic receptors (megalin, cubilin) and apical non-recycling solute carriers (NaPi-IIa, SGLT-2). Defective endocytosis was confirmed by Texas-red-ovalbumin tracing and reduced albumin content. Disruption of Rab-11 and perinuclear galectin-3 compartments suggested mechanistic clues for defective receptor recycling and apical biosynthetic trafficking. p62-dependent autophagy was triggered yet abortive (p62 co-localization with LC3 but not LAMP-1) and PTCs became vacuolated. Impaired lysosomal positioning and blocked autophagy are known causes of cell stress. Thus, early trafficking defects show that Vps34 is a key in vivo component of molecular machineries governing apical vesicular trafficking, thus absorptive function in PTCs. Functional defects underline the essential role of Vps34 for PTC homeostasis and kidney survival

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

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

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.Peer reviewe

Regulation of Macrophage Motility by the Water Channel Aquaporin-1: Crucial Role of M0/M2 Phenotype Switch

The water channel aquaporin-1 (AQP1) promotes migration of many cell types. Although AQP1 is expressed in macrophages, its potential role in macrophage motility, particularly in relation with phenotype polarization, remains unknown. We here addressed these issues in peritoneal macrophages isolated from AQP1-deficient mice, either undifferentiated (M0) or stimulated with LPS to orientate towards pro-inflammatory phenotype (classical macrophage activation; M1). In non-stimulated macrophages, ablation of AQP1 (like inhibition by HgCl2) increased by 2-3 fold spontaneous migration in a Src/PI3K/Rac-dependent manner. This correlated with cell elongation and formation of lamellipodia/ruffles, resulting in membrane lipid and F4/80 recruitment to the leading edge. This indicated that AQP1 normally suppresses migration of resting macrophages, as opposed to other cell types. Resting Aqp1-/- macrophages exhibited CD206 redistribution into ruffles and increased arginase activity like IL4/IL13 (alternative macrophage activation; M2), indicating a M0-M2 shift. In contrast, upon M1 orientation by LPS in vitro or peritoneal inflammation in vivo , migration of Aqp1-/- macrophages was reduced. Taken together, these data indicate that AQP1 oppositely regulates macrophage migration, depending on stimulation or not by LPS, and that macrophage phenotypic and migratory changes may be regulated independently of external cues