IntroductionTumor microenvironment immunity is associated with breast cancer outcome. A high lymphocytic infiltration has been associated with response to neoadjuvant chemotherapy, but the contribution to response and prognosis of immune cell subpopulations profiles in both pre-treated and post-treatment residual tumor is still unclear.MethodsWe analyzed pre- and post-treatment tumor-infiltrating immune cells (CD3, CD4, CD8, CD20, CD68, Foxp3) by immunohistochemistry in a series of 121 breast cancer patients homogeneously treated with neoadjuvant chemotherapy. Immune cell profiles were analyzed and correlated with response and survival.ResultsWe identified three tumor-infiltrating immune cell profiles, which were able to predict pathological complete response (pCR) to neoadjuvant chemotherapy (cluster B: 58%, versus clusters A and C: 7%). A higher infiltration by CD4 lymphocytes was the main factor explaining the occurrence of pCR, and this association was validated in six public genomic datasets. A higher chemotherapy effect on lymphocytic infiltration, including an inversion of CD4/CD8 ratio, was associated with pCR and with better prognosis. Analysis of the immune infiltrate in post-chemotherapy residual tumor identified a profile (cluster Y), mainly characterized by high CD3 and CD68 infiltration, with a worse disease free survival.ConclusionsBreast cancer immune cell subpopulation profiles, determined by immunohistochemistry-based computerized analysis, identify groups of patients characterized by high response (in the pre-treatment setting) and poor prognosis (in the post-treatment setting). Further understanding of the mechanisms underlying the distribution of immune cells and their changes after chemotherapy may contribute to the development of new immune-targeted therapies for breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0488-5) contains supplementary material, which is available to authorized users.

Proteins containing C2 domains are the sensors for Ca 2+ and PI (4,5)P 2 in a myriad of secretory pathways. Here, the use of a freemounting system has enabled us to capture an intermediate state of Ca 2+ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P 2 and IP 3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3-β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P 2 /PI (3,4,5)P 3 , whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI (4,5)P 2 . Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca 2+ -dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.PIP2 | calcium | vesicle fusion C 2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction and in proteins involved in membrane trafficking. They consist of 130 residues and share a common fold composed of two four-stranded β-sheets arranged in a compact β-sandwich connected by surface loops and helices (1-4). Many of these C2 domains have been demonstrated to function in a Ca 2+ -dependent membrane-binding manner and hence act as cellular Ca 2+ sensors. Calcium ions bind in a cupshaped invagination formed by three loops at one tip of the β-sandwich where the coordination spheres for the Ca 2+ ions are incomplete (5-7). This incomplete coordination sphere can be occupied by neutral and anionic (7-9) phospholipids, enabling the C2 domain to dock at the membrane.Previous work in our laboratory has shed light on the 3D structure of the C2 domain of PKCα in complex with both PS and PI(4,5)P 2 simultaneously (10). This revealed an additional lipidbinding site located in the polybasic region formed by β3-β4 strands that preferentially binds to PI(4,5)P 2 (11-15). This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10,(16)(17)(18)(19). Given the importance of PI(4,5)P 2 for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (20-23), it is crucial to understand the molecular mechanisms beneath this event.Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca 2+ and PI(4,5)P 2 combine to orchestrate the vesi...

BackgroundAngiogenesis is a key process for tumor progression and a target for treatment. However, the regulation of breast cancer angiogenesis and its relevance for clinical resistance to antiangiogenic drugs is still incompletely understood. Recent developments on the contribution of microRNA to tumor angiogenesis and on the oncogenic effects of miR-17-92, a miRNA cluster, point to their potential role on breast cancer angiogenesis. The aim of this work was to establish the contribution of miR-20a, a member of miR-17-92 cluster, to tumor angiogenesis in patients with invasive breast carcinoma.MethodsTube-formation in vitro assays with conditioned medium from MCF7 and MDA-MB-231 breast cancer cell lines were performed after transfection with miR-20a and anti-miR20a. For clinical validation of the experimental findings, we performed a retrospective analysis of a series of consecutive breast cancer patients (n = 108) treated with neoadjuvant chemotherapy and with a full characterization of their vessel pattern and expression of angiogenic markers in pre-treatment biopsies. Expression of members of the cluster miR-17-92 and of angiogenic markers was determined by RT-qPCR after RNA purification from FFPE samples.ResultsIn vitro angiogenesis assays with endothelial cells and conditioned media from breast cancer cell lines showed that transfection with anti-miR20a in MDA-MB-231 significantly decreased mean mesh size and total mesh area, while transfection with miR-20a in MCF7 cells increased mean mesh size. MiR-20a angiogenic effects were abrogated by treatment with aflibercept, a VEGF trap. These results were supported by clinical data showing that mir-20a expression was higher in tumors with no estrogen receptor or with more extensive nodal involvement (cN2-3). A higher miR-20a expression was associated with higher mean vessel size (p = 0.015) and with an angiogenic pattern consisting in larger vessels, higher VEGFA expression and presence of glomeruloid microvascular proliferations (p<0.001). This association was independent of tumor subtype and VEGFA expression.ConclusionsTransfection of breast cancer cells with miR-20a induces vascular changes in endothelial tube-formation assays. Expression of miR-20a in breast invasive carcinomas is associated with a distinctive angiogenic pattern consisting in large vessels, anomalous glomeruloid microvascular proliferations and high VEGFA expression. Our results suggest a role for miR-20a in the regulation of breast cancer angiogenesis, and raise the possibility of its use as an angiogenic biomarker.