Gene-expression profiling of microdissected breast cancer microvasculature identifies distinct tumor vascular subtypes

INTRODUCTION: Angiogenesis represents a potential therapeutic target in breast cancer. However, responses to targeted antiangiogenic therapies have been reported to vary among patients. This suggests that the tumor vasculature may be heterogeneous and that an appropriate choice of treatment would require an understanding of these differences. METHODS: To investigate whether and how the breast tumor vasculature varies between individuals, we isolated tumor-associated and matched normal vasculature from 17 breast carcinomas by laser-capture microdissection, and generated gene-expression profiles. Because microvessel density has previously been associated with disease course, tumors with low (n = 9) or high (n = 8) microvessel density were selected for analysis to maximize heterogeneity for this feature. RESULTS: We identified differences between tumor and normal vasculature, and we describe two subtypes present within tumor vasculature. These subtypes exhibit distinct gene-expression signatures that reflect features including hallmarks of vessel maturity. Potential therapeutic targets (MET, ITGAV, and PDGFRβ) are differentially expressed between subtypes. Taking these subtypes into account has allowed us to derive a vascular signature associated with disease outcome. CONCLUSIONS: Our results further support a role for tumor microvasculature in determining disease progression. Overall, this study provides a deeper molecular understanding of the heterogeneity existing within the breast tumor vasculature and opens new avenues toward the improved design and targeting of antiangiogenic therapies

ABCC5 supports osteoclast formation and promotes breast cancer metastasis to bone

INTRODUCTION: Bone is the most common site of breast cancer metastasis, and complications associated with bone metastases can lead to a significantly decreased patient quality of life. Thus, it is essential to gain a better understanding of the molecular mechanisms that underlie the emergence and growth of breast cancer skeletal metastases. METHODS: To search for novel molecular mediators that influence breast cancer bone metastasis, we generated gene-expression profiles from laser-capture microdissected trephine biopsies of both breast cancer bone metastases and independent primary breast tumors that metastasized to bone. Bioinformatics analysis identified genes that are differentially expressed in breast cancer bone metastases compared with primary, bone-metastatic breast tumors. RESULTS: ABCC5, an ATP-dependent transporter, was found to be overexpressed in breast cancer osseous metastases relative to primary breast tumors. In addition, ABCC5 was significantly upregulated in human and mouse breast cancer cell lines with high bone-metastatic potential. Stable knockdown of ABCC5 substantially reduced bone metastatic burden and osteolytic bone destruction in mice. The decrease in osteolysis was further associated with diminished osteoclast numbers in vivo. Finally, conditioned media from breast cancer cells with reduced ABCC5 expression failed to induce in vitro osteoclastogenesis to the same extent as conditioned media from breast cancer cells expressing ABCC5. CONCLUSIONS: Our data suggest that ABCC5 functions as a mediator of breast cancer skeletal metastasis. ABCC5 expression in breast cancer cells is important for efficient osteoclast-mediated bone resorption. Hence, ABCC5 may be a potential therapeutic target for breast cancer bone metastasis

Characterisation of the Stromal Microenvironment in Lobular Breast Cancer

SIMPLE SUMMARY: Invasive lobular breast cancer (ILC) accounts for approximately 5–15% of breast cancers, and although response rates to treatments are initially good, an ILC diagnosis is associated with adverse long-term outcomes; better treatments, specifically targeted to this sub-type of breast cancer, are required to improve patient survival. The tumor microenvironment (TME) plays an important role in determining how cancers respond to treatment, and in this study, we carried out an in-depth analysis of the TME in ILC following laser-capture microdissection of the tumor stroma, and analysis of primary cancer-associated fibroblasts (CAFs), which comprise the majority of non-malignant cells within tumors. This identified changes in genes involved in regulation of the extracellular matrix and also growth factor signaling pathways that were differentially regulated in ILC. Further analysis of breast cancer datasets showed that two of these genes which encode a secreted metalloproteinase (PAPPA) and a metalloproteinase inhibitor (TIMP2) were associated with survival outcomes in ILC. ABSTRACT: Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, and it exhibits a number of clinico-pathological characteristics distinct from the more common invasive ductal carcinoma (IDC). We set out to identify alterations in the tumor microenvironment (TME) of ILC. We used laser-capture microdissection to separate tumor epithelium from stroma in 23 ER+ ILC primary tumors. Gene expression analysis identified 45 genes involved in regulation of the extracellular matrix (ECM) that were enriched in the non-immune stroma of ILC, but not in non-immune stroma from ER+ IDC or normal breast. Of these, 10 were expressed in cancer-associated fibroblasts (CAFs) and were increased in ILC compared to IDC in bulk gene expression datasets, with PAPPA and TIMP2 being associated with better survival in ILC but not IDC. PAPPA, a gene involved in IGF-1 signaling, was the most enriched in the stroma compared to the tumor epithelial compartment in ILC. Analysis of PAPPA- and IGF1-associated genes identified a paracrine signaling pathway, and active PAPP-A was shown to be secreted from primary CAFs. This is the first study to demonstrate molecular differences in the TME between ILC and IDC identifying differences in matrix organization and growth factor signaling pathways

Immunogold localization of photosystems I and II in the green alga Chlamydomonas reinhardtii and the higher plant Pisum sativum : a comparative study

The distribution of Photosystems (PS) I and II was studied in the green alga Chlamydomonas reinhardtii and compared to that found in the higher plant Pisum sativum. Two antibodies were used; one, anti-CP1-e, was raised against the two reaction center proteins of PS I from Synechococcus elongatus, and the other, anti-CP40, was raised against the 40 kDa protein of PS II from the same organism. Immunogold labelling of C. reinhardtii sections showed that both appressed and non-appressed thylakoid membranes were labelled by each antiserum, with labelling on the non-appressed membranes being somewhat (1.7-1.8 times) higher than that on the appressed membranes.In P. sativum, anti-CP1-e labelling was 3.9 times higher on the non-appressed membranes than on the appressed ones, whereas anti-CP40 labelling was equally concentrated on both classes of membranes. The data show that PS distribution in C. reinhardtii is similar to that reported for other algae and different from that in higher plants

Analysis of human histone deacetylase 6 and its associated protein Ubl90

Among mammalian histone deacetylases (HDACs) identified so far, HDAC6 is unique in that it possesses tandem deacetylase domains, as well as a carboxyl-terminal zinc finger motif implicated in ubiquitin binding. HDAC6 has also been demonstrated to deacetylate tubulin and thus potentially contribute to regulation of cell motility. Although HDAC6 has been reported to interact with a variety of nuclear factors, this enzyme is predominantly localized to the cytoplasm.Here I show that the subcellular localization of human HDAC6 is governed by additional elements not present in orthologues of this protein from other species. Human HDAC6 contains a unique insertion of eight or ten repeats of a Ser/Glu-containing tetradecapeptide sequence, here termed the SE14-repeat domain, which is responsible for mediating the Leptomycin B-resistant cytoplasmic retention of human HDAC6. Human HDAC6 also contains a second functional leucine-rich nuclear export signal when compared to murine HDAC6, and a region near the amino terminus of human HDAC6 is capable of mediating nuclear import.I have also identified a previously uncharacterized protein containing a ubiquitin-like domain, here termed Ubl90, as a novel binding partner for HDAC6. Interestingly, Ubl90 binds to the deacetylase domains of HDAC6, rather than to the carboxyl-terminal zinc finger motif previously implicated in ubiquitin association. Ubl90 also interacts with several class I HDACs (HDAC1, HDAC2 and HDAC8), although more weakly than with HDAC6. When Ubl90 is overexpressed, it leads to the redistribution of HDAC1 from a nuclear to a pancellular localization, although this does not affect HDAC1-dependent transcriptional repression in a transient transfection assay. Like HDAC6, Ubl90 is capable of repressing Runx2-mediated transcriptional activation, and, when overexpressed, it leads to cell cycle disturbances similar to those seen with HDAC6 overexpression. These results suggest that Ubl90 and HDAC6 interact functionally, as well as physically

Deficiency of the chromatin regulator Brpf1 causes abnormal brain development

Epigenetic mechanisms are important in different neurological disorders, and one such mechanism is histone acetylation. The multivalent chromatin regulator BRPF1 (bromodomainand plant homeodomain-linked (PHD) zinc finger-containing protein 1) recognizes different epigenetic marks and activates three histone acetyltransferases, so it is both a reader and a co-writer of the epigenetic language. The three histone acetyltransferases are MOZ, MORF, and HBO1, which are also known as lysine acetyltransferase 6A (KAT6A), KAT6B, and KAT7, respectively. The MORF gene is mutated in four neurodevelopmental disorders sharing the characteristic of intellectual disability and frequently displaying callosal agenesis. Here, we report that forebrain-specific inactivation of the mouse Brpf1 gene caused early postnatal lethality, neocortical abnormalities, and partial callosal agenesis. With respect to the control, the mutant forebrain contained fewer Tbr2-positive intermediate neuronal progenitors and displayed aberrant neurogenesis. Molecularly, Brpf1 loss led to decreased transcription of multiple genes, such as Robo3 and Otx1, important for neocortical development. Surprisingly, elevated expression of different Hox genes and various other transcription factors, such as Lhx4, Foxa1, Tbx5, and Twist1, was also observed. These results thus identify an important role of Brpf1 in regulating forebrain development and suggest that it acts as both an activator and a silencer of gene expression in vivo.Peer reviewed: YesNRC publication: Ye

The chromatin regulator Brpf1 regulates embryo development and cell proliferation

With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain-and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity.Wehave recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs.Peer reviewed: YesNRC publication: Ye

Lineage Specification from Prostate Progenitor Cells Requires Gata3-Dependent Mitotic Spindle Orientation

Summary: During prostate development, basal and luminal cell lineages are generated through symmetric and asymmetric divisions of bipotent basal cells. However, the extent to which spindle orientation controls division symmetry or cell fate, and the upstream factors regulating this process, are still elusive. We report that GATA3 is expressed in both prostate basal progenitor and luminal cells and that loss of GATA3 leads to a mislocalization of PRKCZ, resulting in mitotic spindle randomization during progenitor cell division. Inherently proliferative intermediate progenitor cells accumulate, leading to an expansion of the luminal compartment. These defects ultimately result in a loss of tissue polarity and defective branching morphogenesis. We further show that disrupting the interaction between PRKCZ and PARD6B is sufficient to recapitulate the spindle and cell lineage phenotypes. Collectively, these results identify a critical role for GATA3 in prostate lineage specification, and further highlight the importance of regulating spindle orientation for hierarchical cell lineage organization. : In this report, Bouchard and colleagues identify a role for the transcription factor Gata3 in mitotic spindle orientation and lineage specification of prostate progenitor cells through the subcellular localization of the polarity protein aPKC. Their results underscore the importance of mitotic spindle orientation for progenitor cell homeostasis and tissue architecture. Keywords: GATA3, prostate progenitor cells, atypical protein kinase C, aurothiomalate, spindle orientation, prostate development, par complex, cell polarity, lineage specification, epithelial stratificatio