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1.  VAV3 mediates resistance to breast cancer endocrine therapy 
Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process.
A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA–mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression.
The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10−4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy.
This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.
PMCID: PMC4076632  PMID: 24886537
2.  Co-regulated gene expression by oestrogen receptor α and liver receptor homolog-1 is a feature of the oestrogen response in breast cancer cells 
Nucleic Acids Research  2013;41(22):10228-10240.
Oestrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, regulates breast cancer cell proliferation and promotes motility and invasion. To determine the mechanisms of LRH-1 action in breast cancer, we performed gene expression microarray analysis following RNA interference for LRH-1. Interestingly, gene ontology (GO) category enrichment analysis of LRH-1–regulated genes identified oestrogen-responsive genes as the most highly enriched GO categories. Remarkably, chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1 showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα−regulated genes by LRH-1 through binding to oestrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 overexpression stimulated ERα recruitment, while LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at oestrogen response elements controls the expression of oestrogen-responsive genes.
PMCID: PMC3905875  PMID: 24049078
3.  Estrogen receptor splice variants as a potential source of false-positive estrogen receptor status in breast cancer diagnostics 
It is well established that only estrogen receptor (ER)-positive tumors benefit from hormonal therapies. We hypothesized that a subgroup of breast cancer patients expresses estrogen receptor α (ERα), but fails to respond to hormonal therapy due to the expression of a non-functional receptor. We analyzed a series of 2,658 ERα-positive HER2-negative breast tumors for ERα and progesterone receptor (PR) status as determined by mRNA expression and for their molecular subtypes (Luminal type vs Basal type, assessed by BluePrint™ molecular subtyping assay). In addition, we assessed the recurrence risk (low vs high) using the 70-gene MammaPrint™ signature. We found that 55 out of 2,658 (2.1 %) tumors that are ERα positive by mRNA analysis also demonstrate a Basal molecular subtype, indicating that they lack expression of estrogen-responsive genes. These ERα-positive Basal-type tumors express significantly lower levels of both ERα and PR mRNA as compared to Luminal-type tumors (P < 0.0001) and almost invariably (94.5 %) have a high-risk MammaPrint™ profile. Twelve of the MammaPrint™ genes are directly ERα responsive, indicating that MammaPrint™ assesses ERα function in breast cancer without considering ERα mRNA levels. We find a relatively high expression of the dominant negative ERα splice variant ERΔ7 in ERα-positive Basal-type tumors as compared to ERα-positive Luminal-type tumors (P < 0.0001). Expression of the dominant negative ERα variant ERΔ7 provides a rationale as to why tumors are of the Basal molecular subtype while staining ERα positive by immunohistochemistry. These tumors may lack a functional response to estrogen and consequently may not respond to hormonal therapy. Our data indicate that such patients are of MammaPrint™ high recurrence risk and might benefit from adjuvant chemotherapy.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-013-2648-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3742961  PMID: 23912957
Breast cancer; Estrogen receptor variants; Intrinsic subtypes; Molecular subtypes; Tamoxifen
4.  A carrier-assisted ChIP-seq method for estrogen receptor-chromatin interactions from breast cancer core needle biopsy samples 
BMC Genomics  2013;14:232.
The Estrogen Receptor alpha (ERα) is the key transcriptional regulator in luminal breast cancer and is therefore the main target for adjuvant treatment of this subtype. Luminal gene signatures are dictated by the transcriptional capacities of ERα, which are a direct consequence of the receptors binding preference at specific sites on the chromatin. The identification of ERα binding signatures on a genome-wide level has greatly enhanced our understanding of Estrogen Receptor biology in cell lines and tumours, but the technique has its limitations with respect to its applicability in limited amounts of tumour tissue.
Here, we present a refinement of the ChIP-seq procedures to enable transcription factor mapping on limited amounts of tissue culture cells as well as from a limited amount of tumor tissue derived from core needle biopsies. Our approach uses a carrier that can be removed prior to DNA amplification and sequencing.
We illustrate the applicability of this refined technology by mapping the ERα genome-wide chromatin binding landscape in core needle biopsy material from primary breast tumours. With this, our refined technology permits for a high-resolution transcription factor mapping even from clinical samples.
PMCID: PMC3637562  PMID: 23565824
5.  The transcriptional co-factor RIP140 regulates mammary gland development by promoting the generation of key mitogenic signals 
Development (Cambridge, England)  2013;140(5):1079-1089.
Nuclear receptor interacting protein (Nrip1), also known as RIP140, is a co-regulator for nuclear receptors that plays an essential role in ovulation by regulating the expression of the epidermal growth factor-like family of growth factors. Although several studies indicate a role for RIP140 in breast cancer, its role in the development of the mammary gland is unclear. By using RIP140-null and RIP140 transgenic mice, we demonstrate that RIP140 is an essential factor for normal mammary gland development and that it functions by mediating oestrogen signalling. RIP140-null mice exhibit minimal ductal elongation with no side-branching, whereas RIP140-overexpressing mice show increased cell proliferation and ductal branching with age. Tissue recombination experiments demonstrate that RIP140 expression is required in both the mammary epithelial and stromal compartments for ductal elongation during puberty and that loss of RIP140 leads to a catastrophic loss of the mammary epithelium, whereas RIP140 overexpression augments the mammary basal cell population and shifts the progenitor/differentiated cell balance within the luminal cell compartment towards the progenitors. For the first time, we present a genome-wide global view of oestrogen receptor-α (ERα) binding events in the developing mammary gland, which unravels 881 ERα binding sites. Unbiased evaluation of several ERα binding sites for RIP140 co-occupancy reveals selectivity and demonstrates that RIP140 acts as a co-regulator with ERα to regulate directly the expression of amphiregulin (Areg), the progesterone receptor (Pgr) and signal transducer and activator of transcription 5a (Stat5a), factors that influence key mitogenic pathways that regulate normal mammary gland development.
PMCID: PMC3583043  PMID: 23404106
Amphiregulin; Mammary gland; Mammary progenitors; Oestrogen receptor; Progesterone receptor; RIP140; Mouse
6.  Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7–RILP–p150Glued and late endosome positioning 
The Journal of Cell Biology  2009;185(7):1209-1225.
Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150Glued bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)–LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7–RILP complex to remove p150Glued and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.
PMCID: PMC2712958  PMID: 19564404
7.  Activation of endosomal dynein motors by stepwise assembly of Rab7–RILP–p150Glued, ORP1L, and the receptor βlll spectrin 
The Journal of Cell Biology  2007;176(4):459-471.
The small GTPase Rab7 controls late endocytic transport by the minus end–directed motor protein complex dynein–dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP–Rab7–ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150Glued, which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150Glued recruitment by Rab7–RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as βIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150Glued dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7–RILP is transferred by ORP1L to βIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with βIII spectrin, which requires the activities of Rab7–RILP and ORP1L.
PMCID: PMC2063981  PMID: 17283181
8.  Visualizing the action of steroid hormone receptors in living cells  
Transcription controlled by Steroid Hormone Receptors (SHRs) plays a key role in many important physiological processes like organ development, metabolite homeostasis, and response to external stimuli. Understandably, the members of this family have drawn a lot of attention from the scientific community since their discovery, four decades ago. Still, after many years of research we are only beginning to unravel the complex nature of these receptors. The pace at which we do has improved significantly in recent years with the discovery of genetically encoded fluorescent probes, and the accompanying revival of biophysical approaches that allow more detailed study of SHRs. Here, we will look into the different aspects of SHR signalling, and discuss how biophysical techniques have contributed to visualizing their function in their native context, the living cell.
PMCID: PMC1853070  PMID: 17464358
9.  Phosphatidylinositol 4-Kinaseβ Is Critical for Functional Association of rab11 with the Golgi Complex 
Molecular Biology of the Cell  2004;15(4):2038-2047.
Phosphatidylinositol 4-kinaseβ (PI4Kβ) plays an essential role in maintaining the structural integrity of the Golgi complex. In a search for PI4Kβ-interacting proteins, we found that PI4Kβ specifically interacts with the GTP-bound form of the small GTPase rab11. The PI4Kβ-rab11 interaction is of functional significance because inhibition of rab11 binding to PI4Kβ abolished the localization of rab11 to the Golgi complex and significantly inhibited transport of vesicular stomatitis virus G protein from the Golgi complex to the plasma membrane. We propose that a novel function of PI4Kβ is to act as a docking protein for rab11 in the Golgi complex, which is important for biosynthetic membrane transport from the Golgi complex to the plasma membrane.
PMCID: PMC379297  PMID: 14767056
10.  Drug-induced histone eviction from open chromatin contributes to the chemotherapeutic effects of doxorubicin 
Nature Communications  2013;4:1908-.
DNA topoisomerase II inhibitors are a major class of cancer chemotherapeutics, which are thought to eliminate cancer cells by inducing DNA double-strand breaks. Here we identify a novel activity for the anthracycline class of DNA topoisomerase II inhibitors: histone eviction from open chromosomal areas. We show that anthracyclines promote histone eviction irrespective of their ability to induce DNA double-strand breaks. The histone variant H2AX, which is a key component of the DNA damage response, is also evicted by anthracyclines, and H2AX eviction is associated with attenuated DNA repair. Histone eviction deregulates the transcriptome in cancer cells and organs such as the heart, and can drive apoptosis of topoisomerase-negative acute myeloid leukaemia blasts in patients. We define a novel mechanism of action of anthracycline anticancer drugs doxorubicin and daunorubicin on chromatin biology, with important consequences for DNA damage responses, epigenetics, transcription, side effects and cancer therapy.
Anthracycline-based drugs can kill cancer cells by inhibiting topoisomerase II and promoting DNA double-strand breaks. Pang et al. show that anthracyclines also induce eviction of histones from open chromatin regions and, in doing so, modulate DNA repair and apoptosis in human cancer cells.
PMCID: PMC3674280  PMID: 23715267

Results 1-10 (10)